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HK1062033B - Method for labelling and fragmenting dna - Google Patents

Method for labelling and fragmenting dna Download PDF

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Publication number
HK1062033B
HK1062033B HK04105111.9A HK04105111A HK1062033B HK 1062033 B HK1062033 B HK 1062033B HK 04105111 A HK04105111 A HK 04105111A HK 1062033 B HK1062033 B HK 1062033B
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HK
Hong Kong
Prior art keywords
test
measurement
measurements
dna
chemical
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HK04105111.9A
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German (de)
French (fr)
Chinese (zh)
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HK1062033A1 (en
Inventor
Cécile BOURGET
Mitsuharu Kotera
Jean Lhomme
Emmanuelle Trevisiol
Ali Laayoun
Christelle Tora
Isabelle Sothier
Original Assignee
Bio Merieux
Universite Joseph Fourier (Grenoble 1)
Centre National De La Recherche Scientifique
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Priority claimed from FR0106039A external-priority patent/FR2824335A1/en
Application filed by Bio Merieux, Universite Joseph Fourier (Grenoble 1), Centre National De La Recherche Scientifique filed Critical Bio Merieux
Publication of HK1062033A1 publication Critical patent/HK1062033A1/en
Publication of HK1062033B publication Critical patent/HK1062033B/en

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Description

The present invention relates to a DNA fragmentation and marking process and a process for making available probes for detection of a target nucleic acid and the applications of this process particularly in the field of diagnosis.
The state of the art shows that there are many methods for labelling nucleic acids.
The first method is to attach the marker to the base, whether natural or modified; the second method is to attach the marker to sugar, whether natural or modified; and the third method is to attach the marker to phosphate.
Base-based labelling has been used in particular in the approach to nucleic acid labelling by incorporation of directly labeled nucleotides.
Sugar marking is often used in the case of oligonucleotides prepared by chemical synthesis.
Phosphate marking has also been used to introduce functionalised arms and markers during the chemical synthesis of oligonucleotides.
The fact is that the professional, who has to make the marking of a nucleotide, or of a nucleotide analogue or of a nucleic acid, is inclined to make this fixation on the base or on the sugar which offers him more convenience and alternatives.
The fixation of the marker on the phosphate is a more complex technique than the technique of functionalizing the base or sugar and has been used much less often, in particular because of the low reactivity of the phosphate (see for example Jencks W.P. and al J. Amer. Chem. Soc., 82, 1778-1785, 1960).
A second problem arises for the labelling of nucleic acids, especially for large nucleic acids, i.e. more than 100 nucleotides, which must be hybridized with nucleic probes.
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As regards nucleic acid fragmentation, many methods are described in the state of the art.
First, fragmentation can be enzymatic, i.e. nucleic acid fragmentation can be accomplished by nucleases (DNases).
Second, fragmentation can be chemical. For example, in the case of DNA, one can perform DNA depuration or depyrimidination, which is then fragmented in the presence of a base by a mechanism called β-elimination. DNA fragmentation can be accomplished by mechanisms of oxidation, alkylation, addition of free radicals among others.
Finally, fragmentation can be physical, for example by sonication or photochemical.
The difficulty is in actually combining these two stages of fragmentation and marking.
The patent application WO-A-99/65926 describes a process for labeling a synthetic or natural ribonucleic acid (RNA) that involves fragmenting RNA and labeling at the terminal phosphate level. This document describes a number of reactive functions that can be used for labeling in conjunction with fragmentation. These functions allow for labeling of RNAs but a fragmentation step must be associated to have effective labeling because this labeling occurs on the phosphate released during fragmentation. Finally, a significant excess of labeling reagent relative to RNA must be added to achieve effective labeling, which causes background noise problems from the excess DNA in the marking ring.
There is therefore a need for a simple and effective DNA marking technique which allows for good marking efficiency and therefore good sensitivity, which is specific to the marking position and in particular which does not affect the hybridisation properties of the bases involved in the formation of the double helix via hydrogen bonds, and finally which allows for DNA fragmentation in order to hybridise these marked DNA fragments on nuclear probes, and in particular on nuclear probes fixed on a solid medium.
The present invention describes a process for marking and fragmentation of single or double stranded deoxyribonucleic acid (DNA) including: fragment DNA by creating at least one basilic site on that DNA,attach a marker to at least one of the fragments by means of a marking reagent, the marking reagent covalently and majority coupling to at least one phosphate of that fragment, The fragmentation and marking shall be carried out in one step.
The present invention also relates to a process for providing target nucleic acid detection probes, including the marking and fragmentation of single or double stranded deoxyribonucleic acid (DNA) including the following steps: fragment DNA by creating at least one basilic site on that DNA,attach a marker to at least one of the fragments by means of a marking reagent, the marking reagent covalently and overwhelmingly coupling to at least one phosphate of that fragment.
Fragmentation and marking shall be done in one or two steps and marking may be done before, after or simultaneously with fragmentation.
Preferably, the marking and/or fragmentation is carried out in a homogeneous, substantially aqueous solution. Preferably, the marking and fragmentation are carried out simultaneously, i.e. the reagents necessary for these two steps are put together in a substantially aqueous solution with the nucleic acid, for example. This is particularly the case for chemical or enzymatic fragmentation. In the case of mechanical fragmentation by a physical means, marking and fragmentation simultaneously means that the physical means is applied to a solution containing at least the nucleic acids and the marking reagent.
A solution with a significantly aqueous content is a solution containing at least 50% water, preferably containing salts as a buffer solution, and a homogeneous solution is a monophasic solution such as a water/DMSO solution as opposed to a biphasic solution such as a water/chloroform solution.
The fragmentation of DNA by creating a basilic site is done by enzymatic, chemical or physical means.
Chemical DNA fragmentation is performed by introducing nucleic acid into the DNA by a chemical base-site-creation method.
Examples of chemical fragmentation conditions through a basic site of DNA are given in G. Pratviel et al., Angew. Chem. Int. Ed. Engl., 34, p746-769, 1995; G. Pratviel et al., Adv. Inorg. Chem.,45, p251-312, 1998; D. S. Sigman et al. Chem. Rev., 93, p2295-2316, 1993; J. Lhomme et al., Biopolymers (Nucleic Acid Sciences), 52, p65-83, 1999.
A basilic site is created when the N-glycosidic bond linking the modified base to deoxyribose is hydrolyzed, leaving the phosphodiester bond intact. This phenomenon is called depuration (in the case of purines) or depyrimidination (in the case of pyrimidines).
Chemical agents, such as acidic pH, oxidizing agents, or alkylating agents, can induce the phenomenon of depuration and thus the formation of basilic sites. These alkylating agents are electrophilic species that react with the nucleophilic sites of a DNA fragment. The nitrogen atom at position 7 of guanine is the main site of DNA alkylation. In addition to protonation of purines, alkylation is one of the chemical modifications that can make the N-glycoside bond more fragile. - What?
DNA oxidation can also generate so-called alkali-labile basic lesions, which lead to DNA fragmentation in the presence of a base. - What?
The basic site is very unstable, and in its aldehyde form it can easily undergo β-elimination from the phosphodiester fixed at the 3' position, leading to DNA strand fragmentation.
The purification is spontaneous under physiological conditions (pH 7.4 to 37°C) but the reaction rate is very low, in the order of 3.10-11 purifications per second, i.e. unusable for effective fragmentation.
A preferred method of achieving fragmentation is to use an acid pH, i.e. a pH below 5, preferably below 4.
A sodium formate buffer at pH 3 allows the efficient fragmentation of nucleic acids according to the present invention. This buffer is compatible with the one-step marking conditions as will be shown in the examples.
In a particular mode of the present invention, and in order to further increase fragmentation, the deoxyribonucleic acid contains at least one modified base that is likely to generate a base site more easily.
Various modified bases can be used such as N7-alkylpurines, N3-alkylpurines, O6-alkylpurines, 8-bromopurines, 8-thiopurines, 8-alkylthiopurines, 8 azidopurines or 8-alkylsulfonylpurines.
In the case where the nucleic acid to be labelled is generated by an enzyme amplification technique such as PCR, the use of an 8-bromopurine allows for effective incorporation of the nucleotide during amplification, which facilitates the process of fragmentation and labelling according to the invention, while maintaining excellent sensitivity for the enzymatic amplification step. An 8-methylthiopurine is also incorporated without interfering with the efficiency of amplification and incorporation by PCR. Once incorporated, the thioether base is hydrolyzed by a peracid or monopulver derivative such as potassium peroxymulfate of 1000 hours, for example in sulphonylurea, which has been shown to be very effective.
A marker is at least one marker capable of generating a detectable signal directly or indirectly. enzymes that produce a signal detectable e.g. by colorimetry, fluorescence, luminescence, such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose-6-phosphate dehydrogenase,chromophores such as fluorescent compounds, luminescent dyes,electron density groups detectable by electron microscopy or by their electrical properties such as conductivity, amperometry, voltammetry, impedance,e.g. detectable groups, e.g. molecules of sufficient size to induce detectable changes in their physical and/or chemical characteristics, this detection can be achieved by methods such as the optical resonance, the surface angle of the plasma, the radiation or the physical variation of the surface of the molecules, the radiation or the radiation, the physical effect, the radiation or the physical effect of the atoms, the radiation or the radiation, the radiation or the physical effect of the atoms, the radiation or the radiation, the radiation or the radiation, the radiation or the radiation, the radiation or the radiation, the radiation or the radiation, the radiation or the radiation, the radiation, the radiation or the radiation, the radiation, the radiation or the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the radiation, the
Preferably, the marker is not a radioactive marker to avoid safety problems associated with such markers.
In a particular embodiment of the present invention, the marker is electrochemically detectable and in particular the marker is a derivative of an iron complex such as a ferrocene.
Indirect systems can also be used, such as ligands capable of reacting with an anti-ligand. Ligand/anti-ligand pairs are well known to the professional, such as biotin/streptavidine, haptene/antibody, antigen/antibody, peptide/antibody, sugar/lectin, polynucleotide/complementary polynucleotide. In this case, the ligand carries the reactive function. The anti-ligand may be directly detectable by the markers described in the previous paragraph or be detectable by another ligand/anti-ligand pair.
Another example of indirect systems uses a specific covalent bond between the ligand and the antiligand, e.g. methyl ketone and alcoholxyamine.
In the process of the present invention, the bond between the marking reagent and the nucleic acid is covalent, but it is described above that non-covalent interactions may be used in particular in stacking systems or in the case where the marker is detectable indirectly.
Such indirect detection systems may, under certain conditions, lead to signal amplification and reference may be made to the applicant's earlier patent applications WO-A-00/07982, WO-A-01/92361 and WO-A-95/08000 for examples of chemical amplification using polymers or to the applicant's application WO-A-01/44506, also for stack chemical amplification systems.
In a particular signal amplification mode, at least two markers are present on the marking reagent.
In a preferred mode of the invention, the tracer is a low-sterile fluorescent compound such as fluorescein, danzyl, IR-type chromophores (Li-COR Inc, Lincoln NE, USA), cyanine derivatives such as Cy5 and Cy3 (Randolph J.B. and al, Nucleic Acids Res., 25(14), p2923-2929, 1997) and in particular Cy5 derivatives or the tracer is a low-sterile haptene such as biotin or a derivative of abi-etane (see application WO-A-00/07982).
In the case of a fluorophore, it is preferable to work with fluorophores with an excitation wavelength greater than 450 nm, preferably greater than 600 nm.
In the case of the tracer being a hapten that does not produce a signal on its own, such as biotin, the detection is carried out by the reaction of an anti-ligand as described above. In the case of biotin, preferably streptavidine or an anti-biotin antibody is used in combination with a fluorescent compound such as fluorescein, Cy5 or Phycoerythrin. In the case of abietane, a monoclonal antibody is used as described in WO-A-00/07982.
The term deoxyribonucleic acid or DNA means a chain of at least two deoxyribonucleotides possibly including at least one modified nucleotide, for example at least one nucleotide with a modified base such as inosin, methyl-5-deoxycytidine, dimethylamino-5-deoxyuridine, deoxyuridine, diamin-2,6-purine, bromo-5-deoxyuridine or any other modified base allowing hybridisation.
DNA can also be altered at the internucleotide level such as phosphorothioates, H-phosphonates, alkyl-phosphonates, at the skeletal level such as alpha-oligonucleotides (FR-A-2.607.507) or PNAs (M. Egholm et al., J. Am. Chem. Soc., 114, 1895-1897, 1992).
In particular, DNA is obtained by an enzyme amplification technique such as: the Polymerase Chain Reaction (PCR) described in US-A-4,683,195, US-A-4,683,202 and US-A-4,800,159 and its RT-PCR (Reverse Transcription-PCR) derivative, including in a one-step format as described in patent application EP-A-0.569.272, the Ligase Chain Reaction (LCR) described for example in patent application EP-A-0.201.184, and the Repair Chain Reaction (RCR) described in patent application WO-A-90/01069.
Amplions are then used to refer to DNA generated by an enzyme amplification technique, and may also include ribonucleotides in a small proportion, for example less than 10%.
Each of these changes can be taken in combination as long as at least one phosphate is present in the DNA.
The labelling reagent shall include, as the reactive function, a pattern selected from the following compounds: diazomethyl; alkyl halides; nitrosocures; spirocyclopropane; aziridine; epoxides; trifluorosulfonates.
The reactive functions are described below:
For the alkyl halogenide reactive function, X stands for Br, Cl or I. The advantageous marking reagent is 5-bromomethyl) fluorescein.
For the nitroso acid reactive function, R5 is an alkyl or H.
The diazomethyl function has already been used for alkylation of phosphate groups, but a number of problems arise: on the one hand, diazo derivatives in general are unstable themselves, which generates problems for the use of these labelling reagents in a labelling kit, and on the other hand, the coupling product is unstable which is redundant if the labelled product has the function of highlighting the presence of a biological target molecule in any sample, or if it is the labelled target that is to be detected.
Finally, the derivatives with diazomethyl function are insoluble in water, which leads to the use of biphasic conditions for coupling with nucleic acids, which are soluble and stable only in water or aqueous buffers, but these conditions slow down the reaction rate and thus impair the coupling efficiency.
In a preferred process mode of the invention, the marking reagent is selected from the compounds of formula (1): in which: R1 represents H or an alkyl, alkyl-substituted, aryl or aryl-substituted group,Z includes a detectable marker. Z and/or R1 are chosen to stabilize the diazomethyl function, i.e. at least one of the two Z or R1a groups has a phenyl nucleus.
Preferably, the labelling reagent shall be selected from the compounds of formula (2): - What? in which: R1 represents H or an alkyl, aryl or substitute aryl group,R2 is a detectable marker,L is a bonding arm with a linear chain of at least two covalent bonds and n is equal to 0 or 1,etZ is chosen from: - What? in which: ■ R3 and R4 are represented independently of each other: H, NO2, Cl, Br, F, I, OR, SR, NR2, R, NHCOR, CONHR, COOR with R = alkyl or aryl, ■ -Y-X- represents -CONH-, -NHCO-, -CH2O-, -CH2S-.
In a particular mode according to formula (1), Z has the following structure: In this case and if R1 is equal to H, the labelling reagent is 1-Pyrenyldiazomethane (PDAM).
Although this marker is fluorescent, the excitation wavelength is too close to that of nucleic acids. Indirect detection by the use of a monoclonal antibody directed against the pyrene pattern is preferred. The method of obtaining this antibody is well known to the professional (see for example patent application WO-A-00/07982).
In a preferred method of manufacture, the labelling reagent is of the formula (3): - What? in which: R1 represents H or an alkyl, aryl or aryl group substituted,R2 represents a detectable marker,L is a bonding arm with a linear sequence of at least two covalent bonds and n an integer equal to 0 or 1,R3 and R4 represent independently of each other: H, NO2, Cl, Br, F, I, OR, SR, NR2, R, NHCOR, CONHR, COOR with R = alkyl or aryl,-Y-X- represents -CONCHH-, -NHCO-, -CH2O-, -2S-.
The reagent is of the formula (4): in which: R1 represents H or an alkyl, aryl or aryl group substituted,R2 represents a detectable marker,L is a bonding arm with a linear sequence of at least two covalent bonds and n an integer equal to 0 or 1,R3 and R4 represent independently of each other: H, NO2, Cl, Br, F, I, OR, SR, NR2, R, NHCOR, CONHR, COOR with R = alkyl or aryl.
The advantage is that the reagent has the formula (5): - What? in which: R1 represents H or an alkyl, aryl or aryl group substituted,R2 represents a detectable marker,L is a bonding arm with a linear sequence of at least two covalent bonds and n an integer equal to 0 or 1,R3 and R4 represent independently of each other: H, NO2, Cl, Br, F, I, OR, SR, NR2, R, NHCOR, CONHR, COOR with R = alkyl or aryl.
The reagent is of the formula (6): - What? in which: R1 represents H or an alkyl, aryl or aryl group substituted,R2 represents a detectable marker,L is a bonding arm with a linear sequence of at least two covalent bonds and n an integer equal to 0 or 1,R3 and R4 represent independently of each other: H, NO2, Cl, Br, F, I, OR, SR, NR2, R, NHCOR, CONHR, COOR with R = alkyl or aryl.
In the above formulae (3) to (6), R3 and R4 are advantageously represented independently of each other: H, NO2, OCH3.
Thus, a compound preferred according to formula (6) is of formula (6'): - What? in which: R1 represents H or an alkyl, aryl or aryl group substituted,R2 represents a detectable marker,L is a bonding arm with a linear chain of at least two covalent bonds and n an integer equal to 0 or 1.
A compound preferred according to formula (4) is of formula (4'): - What? in which: R1 represents H or an alkyl, aryl or aryl group substituted,R2 represents a detectable marker,L is a bonding arm with a linear chain of at least two covalent bonds and n an integer equal to 0 or 1.
In a particular mode of the process where the signal is to be amplified, at least two markers are present on the marking reagent. - What? in which: R2 represents a detectable marker,m is an integer between 1 and 100, preferably between 1 and 20,p is an integer between 1 and 10, preferably 2 to 6, preferably 4.
This structure of R2-(L) n applies equally to the previous formulae (2) to (6).
Another preferred marking reagent for signal amplification is the reagent of formula (8): - What? in which: R2 is a detectable marker,R3 is H, NO2, Cl, Br, F, I, OR, SR, NR2, R, NHCOR, CONHR, COOR with R = alkyl or aryl,L is a bonding arm with a linear chain of at least two covalent bonds and n is an integer equal to 0 or 1,-Y-X- is -CONH-, -NHCO-, -CH2O-, -CH2S-.
The reagent for signal amplification is given by the formula (9) - What? in which: R2 represents a detectable marker,R3 represents H, NO2, Cl, Br, F, I, OR, SR, NR2, R, NHCOR, CONHR, COOR with R = alkyl or aryl, preferably R3 represents H, NO2 or OCH3.,L is a bonding arm with a linear chain of at least two covalent bonds and n an integer equal to 0 or 1.
Some of the advantageous reagents of the invention are: (a) of formula (10): - What? in which: R1 represents H or an alkyl group or aryl or aryl substituent,R2 represents a detectable marker,L is a bonding arm with a linear sequence of at least two covalent bonds and n an integer equal to 0 or 1,b) of formula (11): in which: R1 represents H or an alkyl group or aryl or aryl substituent,R2 represents a detectable marker,L is a bonding arm with a linear sequence of at least two covalent bonds and n an integer equal to 0 or 1,c) of formula (12): - What? where:R1 represents H or an alkyl or aryl or aryl substituent group,R2 represents a detectable marker,L is a bonding arm with a linear chain of at least two covalent bonds and n an integer equal to 0 or 1.
The labelling reagent shall preferably have: (a) the structure - What? where R1 represents a methyl group or a phenyl, and - What? where R1 represents a methyl or phenyl group, or - What? where R1 represents a methyl or phenyl group.
Other preferred reagents according to the invention have the formula (13): - What? in which: R2 represents a detectable marker,L is a bonding arm with a linear sequence of at least two covalent bonds and n an integer of 0 or 1.
In particular, the labelling reagents according to the method of the invention are soluble in polar water-soluble solvents such as DMF, DMSO, CH3CN, THF, DMA (dimethylacetamide), NMP (N-methylpyrrolidone), DME (dimethoxyethane).
Preferably, the labelling reagents are soluble in DMSO or water.
Water-miscible solvent means a solvent that is miscible in a proportion of at least 5% by volume with water or an aqueous buffer containing salts.
The L-arm is advantageous in the previous formulae, as it includes an ethylene glycol or polyethylene glycol pattern to increase the solubility of the reagent in water.
These reagents can thus be homogeneously bound to nucleic acids, the homogeneous phase being a substantially aqueous solution, i.e. containing at least 50% water.
One of the purposes of the present invention is to describe a method for detecting single or double stranded target deoxyribonucleic acid (DNA) using the following steps: (a) fragment and mark the DNA in any of the above described ways; (b) hybridise the marked fragments on at least one sufficiently specific nucleic probe of the target nucleic acid; (c) detect the hybrid formed by means of the marker.
As will be shown in the examples, denaturing DNA before hybridization increases sensitivity.
In a particular mode of the process, an enzymatic amplification step takes place before fragmentation and marking. The enzymatic amplification step generates DNA amplifiers from a target DNA nucleic acid, but also from a target RNA nucleic acid such as messenger RNA, transfer RNA or ribosomal RNA.
Preferably, the fragmentation, marking and denaturation steps take place simultaneously, both in the case of a natural target nucleic acid and in the case of a target nucleic acid obtained by an enzymatic amplification technique.
Fragmentation by the creation of a basic site involves the loss of a base. In target nucleic acid detection processes and particularly in genotyping processes, i.e. in processes where the target nucleic acid has a polymorphism spread over its sequence, it is necessary to identify a plurality of sequence modifications, some of which represent the change of a single base between the target and the nucleic probes whose function is to identify this change (capture probes). Specificity is therefore essential in this context.
The present invention also concerns a method for detecting polymorphism, whether or not distributed in predetermined positions of a target nucleic acid by the presence of a plurality of deletions and/or insertions and/or mutations in the sequence of the target nucleic acid relative to a sequence, called a reference, involving the following steps: (a) have a target DNA containing all the polymorphism to be studied, such DNA possibly generated by an enzymatic amplification technique, (b) fragment and mark such DNA by one of the processes described above, (c) hybridise such fragments on a plurality of nucleic probes called capture probes, the plurality of capture probes being fixed on a solid support and the plurality of capture probes covering as a whole at least the polymorphism to be studied, (d) detect the hybrids formed between the marism fragments and at least part of the nucleic probes by means of the marking and deduce the polymorphism of the DNA from the marking.
As mentioned above, a denaturation step after the fragmentation and marking step improves the sensitivity of the process, and this step is preferably carried out simultaneously with the marking and fragmentation step.
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The invention allows the use of a marked DNA, as defined above, as a probe for detecting a target nucleic acid.
To allow the detection and/or quantification and/or purification of the target nucleic acid, the tagged DNA is capable of forming a hybridisation complex with a nucleic probe.
The detection method is applicable to the sequencing, expression profile of messenger RNAs or mutation screening for research purposes as well as drug screening in the pharmaceutical industry, diagnosis of infectious diseases (including bacteriology, virology, parasitology) or genetics, food or industrial control.
Many publications describe this type of application: Antoine de Saizieu et al, Nature Biotechnology, 16, p44-48, 1998; Thomas R. Gingeras et al Genome Research, 8, p435-448, 1998; David J. Lockhart et al, Nature Biotechnology, 14, p1675-1680, 1996; Daniel D. Shoemaker et al, Nature Genetics, vol 14, p450-456, 1996; R.J. Lipshutz et al; BioTechniques, 19(3), p442-447,1995; David G. Wang et al, Science, 280, p1077-1082, 1998; L. Gunderson et al; Genome Research, 8, p1142-1152, 1998; Kevin Joseph G. Hacia and al; Nature Genetics, vol 14, p441-447, 1996. This is the first time that the use of this method has been reported in the literature.
The trend in diagnostics and particularly for infectious diseases (e.g. AIDS or Tuberculosis) is to lower the sensitivity level to the detection of a single molecule in a sample, which may be several milliliters in the case of a sample of blood or urine or cerebrospinal fluid. This level of sensitivity can only be achieved if all steps from sample collection to the required result are used. In particular, where an optimized enzymatic amplification step is required to obtain the necessary sensitivity (viral or bacterial infection with HIV, HCV or Tuberculosis), a marking and/or fragmentation process as described in the present invention does not affect sensitivity, as this technique does not replace the deoxyribonucleotide de-amplification technique, because it does not incorporate the enzyme deoxyribonucleotide sensitivity, as this technique does not replace the deoxyribonucleotide de-amplification technique.
Further information can be found in another patent application by Applicant FR-2 824 323 filed on 4 May 2001 under registration number FR01/06040 and its international extension filed on the same day as the present invention.
The figures and examples given herein represent particular embodiments and cannot be considered as limiting the scope of the present invention. Figure 1 shows the developed formulae of different reagents used in the present invention and the abbreviation for them (o- stands for ortho, m-meta and p-para).Figures 2A to 2I show the time-dependent capillary electrophoresis profiles of the covalent coupling of different reagents having a diazomethyl function on the 3'-monophosphate (3'-UMP) uridine as shown in example 6.1. The test chemical is used to determine the concentration of the test chemical in the test medium.DPDAM at 20 mM in Figure 2C,PMDAM in Figure 2D,NPDAM in Figure 2E,BioDPDAM in Figure 2F,meta-BioPMDAM in Figure 2G,para-BioPMDAM in Figure 2H, etortho-BioPMDAM in Figure 21.Figure 3A to 3D represents the time-based profiles analyzed by capillary electrophoresis of the reaction of meta-BioPMDAM on four (4) 3'-monophosphate nucleotides as shown in Example 6.2. The molecules are as follows: 3'-CMP in ribonucleotide series according to Figure 3A,3'-AMP in ribonucleotide series according to Figure 3B,3'-GMP in ribonucleotide series according to Figure 3C, and3'-TMP in deoxyribonucleotide series according to Figure 3D.Figure 4 represents the time-based profiles analysed by capillary electrophoresis of the reaction of meta-BioPMDAM on a 5'-ApUp dinucleotide according to Example 6.3.Figures 5A to 5D represent the proton NMR spectrum in D2O of the various conjugates between the meta-BioPMDAM reagent and four (4) 3'-monophosphate ribonucleotides as shown in example 6.4. 3'-GMP in Figure 5A,3'-AMP in Figure 5B,3'CMP in Figure 5C, and3'UMP in Figure 5D.Figure 6 shows a synthesis scheme of a two-biotin-containing marker reagent for chemical signal amplification.Figure 7 shows the mechanism of acidic fragmentation by basal site formation.Figure 8 shows, as shown in Example 8.1, the acid pH degradation kinetics for different modified nucleosides (8-bromo-2'-deoxyadenosine (8-BrdA) and 5-bromo-2'-deoxytidine (5-BrdC)) as well as the four natural nucleosides (dA,The results are represented as percentage of the starting nucleoside hydrolysis (in order) relative to the reaction time in minutes (in abscissa).Figure 9 represents, in Example 11.2, the time-related marking kinetics at 60°C with the PDAM reagent on a synthetic 5'-phosphate ODN.The results are represented as percentage of marking relative to the reaction time in minutes (in abscissa).Figure 10 represents, in Example 11.3, the percentage of marking relative to the reaction temperature.The results are presented in Figure 10 with the time-related marking percentage and in abscissa.Figure 11 shows a synthesis pathway for a reagent with formula 4', using the commercial reagent 5-nitrovanillin.
Example 1 - Synthesis of reagents with biotin: General summary scheme:
Example 1.1: synthesis of the meta-BioPMDAM: • Compound biotin methacetophenone 1a:
D-biotin (1,0 g, 4,1 mmol) is dissolved in 45 ml of anhydrous DMF on hot heat. It is cooled to 0 °C under argon, then N-methylmorpholine (590 microlitres (μL), 5,33 mmol) and isobutyl chloroformate (840 μL, 6,60 mmol) are added successively. It is stirred for 30 minutes (mn), then 3-aminoacetophenone (824 mg, 6,10 mmol) and N-methylmorpholine (480 L, 4,35 mmol) are added to 10 ml of DMF. The solution is stirred at 0 °C for 2 hours (h), then evaporated to dry. The remaining powder is added to 3 g of MeOHOH, then added to 1 g of MeOH. The product is given as a white powder, then 1 g of CH2O2 (OH) is added to 70 g of MeOH, and then given to the water to be filtered.
The measurement of the radiation emitted from the gas turbine is carried out at a temperature of approximately -15 °C (~15 °C) and a temperature of approximately -15 °C (~15 °C).
• Compound methahydrazone 2a:
A solution of 1a (500 mg, 1.38 mmol) and hydrazine monohydrate (200 μL, 4.15 mmol) in absolute ethanol (8 mL) is heated at low temperature for 2 h. After cooling to room temperature, the white precipitate is filtered, washed with water, then with ether and dried, resulting in 385 mg (74%) of product 2a as a white powder.
The measurement of the radiation emitted from the gas turbine is based on the following equation:
• Compound metadiazomethane 3a:
2a (180 mg, 0.48 mmol) is solubilised in 2 mL of DMF. MnO2 (340 mg, 3.9 mmol) is then added. After 30 minutes of stirring at room temperature, the mixture is filtered through a fritted funnel containing cellite (thickness: 0.5 cm) and molecular sieves in powder form 3 Å (0.5 cm). The reaction mixture is concentrated to a volume of about 0.5 mL, then 5 mL of ether is added. The resulting precipitate is filtered, washed with ether and then dried.
The temperature of the water is measured at a temperature of approximately 160 °C. - IR (KBr) : 3278, 2935, 2859, 2038, 1704, 1666, 1605, 1577, 1536, 1458, 1430, 1263 cm-1. - RMN 1H (300 MHz) δ = 1,3-1,7 (m, 6 H) ; 2,11 (s, 3 H); 2,28 (t, J= 8 Hz, 2 H); 2,57 ; (d, J= 12 Hz, 1 H) ; 2,81 (dd, J= 12 and 5 Hz, 1 H) ; 3,11 (m, 1 H) ; 4,13 (m, 1 H) ; 4,29 (m, 1 H) ; 6,33 (s, 1 H) ; 6,41 (s, 1 H) 6,60 (m, 1 H) ; 7,25 (m, 3 H) ; 9,84 (s, 1 H).
Example 1.2: synthesis of the para-BioPMDAM: • Compound biotin paraacetophenone 1b:
The solution is maintained at 0°C for 2 h and then evaporated to dry. The residue is taken up again in 50 mL of water. The resulting precipitate is filtered, washed with water and then with 50 mL of MeOH dissolved. The white precipitate is obtained in DMF by evaporating the solution and then washed to 60 mg MeOH and evaporated to 88 mmol (246 mmol, 1 mL).
The measurement of the radiation emitted from the gas turbine shall be carried out at a temperature of ± 5 °C and a pressure of ± 5 °C.
• Parahydrazone 2b compound:
The solution is heated at low temperature for 3 hours. The resulting white precipitate is filtered, washed with ice water, and 820 mg (90%) of product 2b is obtained as a white powder.
The following is the list of the substances which are to be used in the preparation of the product:
• Compound para-diazomethane 3b:
2b (200 mg, 0.53 mmol) is solubilised in 10 mL of DMF. 800 mg of MnO2 is then added. After 10 minutes of stirring, the mixture is filtered through a cellular (0.5 cm) -molecular (0.5 cm) sieve. The reaction mixture is evaporated dry and then washed with ether and dried.
The measurement of the radiation emitted from the gas turbine is carried out at a temperature of approximately 180 °C (dec). - IR (KBr): 3257, 2930, 2857, 2032, 1698, 1597, 1524, 1510, 1455, 1404, 1307, 1259, 1180 cm-1. - RMN 1H (200 MHz, DMSO-d6) δ = 10,18 (s, 1H, NH-CO) ; 7,88 (d, 2H, J= 6 Hz, Ax-H) ; 7,7 (d, 2H, J= 6 Hz, Ar-H) ; 6,41 (s wide, 1H, NH-CO-NH) ; 6,34 (s wide, 1 H, NH-CO-NH) ; 4,28 (m, 1H, CH2-CH-NH) ; 4,12 (m, 1H, CH-CH-NH) ; 3,11 (m, 1H, CH-SJA) ; 2,80 and 2,55 (s system; ABX, 2H, CH-CH= 2H, 2J, 5J, 3H, CH= 2H, CH= 2H, 2H, CH= 2,60; 3H, CH= 2H, CH= 2H, 2H, CH= 2,35 (m, 2H, CH-CH-CH-CH) ; 2,40 (m, 2H, 2H, CH= 2H, 2H, 2H, CH= 2H, 2H, 2H, CH= 2H, 2H, 2H, 3H, 2H, CH-CH-CH-CH) ; 3,0); 3,0 (m, 2H, 2H, 2H, 2H, 2H, 2H, CH= 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2
Example 1.3: Synthesis of the ortho-BioPMDAM: • Compound biotin orthoacetophenone 1c:
The solution is kept agitated at room temperature for 1.1 to 3 hours and evaporated dry. The residue is cooled to 0°C under argon and then N-methylmorpholine (590 μL, 5.33 mmol) and isobutyl chloroformate (840 μL, 6.60 mmol) are added successively. The solution is agitated for 30 min, then 2-aminoacetophenol (824 mg, 6.10 mmol) is added. The solution is kept agitated at room temperature for 1.1 to 3 hours and 30 minutes, then evaporated dry. The residue is precipitated in 50 mL of water. The resulting precipitate is filtered, the raw water is dissolved with 50 mL of MeOH at high temperature. The resulting precipitate is filtered, dissolved with water.
The following is the list of the substances which are to be used in the preparation of the product: - F 150°C - IR (KBr) : 3248, 2930, 2857, 2359, 1691, 1669, 1651, 1582, 1528, 1448, 1354, 1310, 1245, 1161 cm-1. - RMN 1H (200 MHz, DMSO-d6) δ = 11,24 (s, 1H, NH-CO) ; 8,33 (d, 1H, J = 8,5 Hz, Ar-H) ; 7,97 (d, 2H, J = 8 Hz, Ar-H) ; 7,57 (t, 1H, J= 7 Hz, Ar-H) ; 7,18 (t, 1H, J = 7 Hz, Ar-H) ; 6,44 (s wide, 1H, NH-CO-NH) ; 6,35 (s wide, 1H, NH-CO-NH) ; 4,30 (s system, 1H, 2H-CH-NH) ; 4,14 (s CH, 1H, 1H-H) ; 3,12 (s 2,12), 3H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H, 2H
• Compound orthohydrazone 2c:
The 1c compound (500 mg, 1.38 mmol) is heated and dissolved in ethanol (99% 8 mL) and then hydrazine monohydrate (572 μL, 11.1 mmol) is added. The solution is heated at low temperature for 50 minutes. The solution is evaporated dry. The resulting white precipitate is filtered, washed with water and then dried with ether.
The following is the list of the substances which are to be used in the preparation of the additive: fats, fats and oils, and mixtures of these products:
• Compound ortho-diazomethane 3c:
2c (200 mg, 0.53 mmol) is solubilised in 10 mL of DMF. 800 mg of MnO2 is then added. After 15 minutes of stirring, the mixture is filtered through a mixture of Cellite (0.5 cm) -Tamis (0.5 cm) molecular powder. The reaction mixture is evaporated dry and then washed with ether and dried.
The following is the list of the active substances which are to be used in the preparation of the active substance:
Example 1.4: synthesis of the meta-BioDPDAM: • Compound metabenzophenone 1d:
Solubilize D-biotin (500 mg, 2.05 mmol) in 23 mL of anhydrous DMF on hot water. Cool to 0°C under argon, then add successively N-methylmorpholine (295 μL, 2.67 mmol) and isobutyl chloroformate (420 μL, 3.28 mmol). Agitate for 30 min, then add 3-aminobenzophenone (605 mg, 3.07 mmol) and N-methylmorpholine (240 μL, 2.17 mmol) to 7 mL of DMF. Agitate the solution at 0°C for 2 h, then dry evaporate. The residue is collected in 1 mL of MeOH, then added 25 mL of evaporated water. The resulting solution is filtered, precipitated in water to give 72% (92 mg) of MeOH, then recrystallized with 1 mg (130 mg) of MeOH.
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• Compound methahydrazone 2d:
The solution is heated at reflux for one night. After evaporation, the product is recrystallized in 1 mL of ethanol and water. The white precipitate is recrystallized: it is dissolved in a minimum of hot ethanol and water is added until a slight turbidity appears. After cooling, the precipitate obtained is washed with water and then etherised.
The measurement of the radiation emitted from the air is based on the following equations:
• Compound metadiazodiphenyl 3d:
3d (500 mg, 0.53 mmol) is solubilised in 1 mL of THF. 80 mg of activated MnO2 is then added. After 5 minutes of stirring at room temperature, the mixture is filtered through a cellite (0.5 cm) -molecular thymus (0.5 cm) mixed layer. The reaction mixture is evaporated dry.
The measurement of the emission of CO2 from the combustion of the fuel is carried out by the following methods:
Example 2 : Marking reagent with Cy5 : Cy5-PMDAM:
• Compound Iodide of 2-[4-[N-acetyl-N-phenylamino) but-1-dienylyl-1,2,3-tetramethyl[3H] indolium 6
The mixture of malonaldehyde-bis (phenyl) monochloride 5 (18.3 g; 70.0 mmole), NaOAc (9.0 g; 110 mmole) and 1,2,3,3-tetramethyl[3H]indolium iodide 4 (4.25 g; 14.1 mmole) in acetic anhydride (75 mL) is heated to 110 °C (70 °F) for 20 min. After cooling, ether (350 mL) is added and the precipitated brown solid is filtered, washed with ether (3 x 100 mL).
The following are the values of the 1H (CDCl3) NEM: δ = 8,64 (d ; 1H ; J = 12 Hz ; 1-H) ; 8,14 (t ; 1H ; J = 16 ; 12 Hz ; 3-H) ; 7,63-7,19 (m ; 9H) ; 6,90 (d ; 1H ; J = 15 Hz ; 4-H) ; 5,82 (t ; 1H ; J= 12 ; 13 Hz ; 2-H) ; 4,06 (s ; 3H ; NCH3) ; (2 ,16 (s ; 3H ; -COCH3) ; 1,74 (s ; 6H ; CH3).
•Compound Bromide of 1- ((5-carboxypentyl)-2,3,3-trimethyl[3H]indolium 9 with:
The 2,3,3-trimethylindole 7 (10.0 g; 62.8 mmole) and 6-bromohexanoic acid 8 (12.3 g; 62.8 mmole) are mixed without solvent and heated at 110°C for 12 h under argon. The red-violet paste reaction mixture is washed with ethyl acetate (2 x 60 mL, crushed with spatula and settle the surfactant), then with acetone (50 mL, the paste solidifies). The pink solid is filtered and then emptied under 73 g (16.0 %).
• Compound Cy5COOH 10:
The mixture of iodide 6 (6.0 g; 12.7 mmol), bromide 9 (4.5 g; 12.7 mmol) and NaOAc (2.6 g; 32 mmol) in acetic anhydride (35 mL) is heated to 110 °C for 20 min. After cooling, ether (150 mL) is added and the precipitate is filtered and washed with ether (3 × 50 mL). The solid is dissolved in 100 mL of CH2Cl2, filtered and purified by SiO2 column chromatography (elevant: MeOH 5-10%/CH2Cl2).
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• Compound of 3-aminoacetophenone coupled with Cy5COOH 10 (product 11)
After 5 minutes of stirring, 3-aminoacetophenone (488 mg; 3.6 mmole) is added. The mixture is stirred at room temperature for 3 h. 250 mL of ether is added to produce a paste solid. After stirring, the solid is allowed to rest on the bottom of the balloon and dried under drying. 50 mL of isobutyl chlorophosphate is added again and evaporated with a spatula to obtain a solid. The liquid is filtered with water, then extracted from the liquid (Other) and passed to the column of ether (Iranium-Cl) to obtain a 0.7 g of ether.
• Cy5-hydrazone 12 compound:
Add hydrazine monohydrate (180 μL; 3.1 μmol) to a solution of acetophenone 11 (0.93 g; 1.46 mmole) in 5 mL of absolute ethanol and stir at room temperature for 7 h. Add 50 mL of ether and filter the precipitate and wash with ether. Dissolve the raw product in 50 mL of CH2Cl2, then filter the solution to 10 mL. Precipitate the product by adding 100 mL of ether, filter, ether wash and vacuum dry.
• Cy5PMDAM 12bis compound:
A solution of 100 mg hydrazone 12 in 2 mL DMF is added 300 mg MnO2 and stirred vigorously for 10 min. The suspension is filtered through a layer of cellulite and washed with DMF (3×500 μL). 50 mL ether is added and the deposited oil is crushed with a spatula and the surfactant is settled. The washing operation is repeated three times with 25 mL ether and the resulting solid is filtered, dried. 65 mg (65%) 12bis product is obtained. The purity of the product is about 80-85% (RMN 1H).
Example 3: Other synthesized reagents: The following is a list of the active substances in the active substance:
4-nitrobenzaldehyde hydrazone is commercially available (reference 28,118-2, Aldrich, France). The solution is left to agitate for 5 minutes and then 1.26 g (4 equivalent, 14.56 mmole) of MnO2 is added carefully. The mixture is left to agitate for 10 minutes and then filtered. The recovered filtrate is evaporated dry. After washing with pentane, the compound para-nitrophenyldiazomethane is obtained as a bright orange powder with an efficiency of 79% (468 mg, 2.87 mmole).
The measurement of the radiation is based on the following equation:
Example 3.2: Synthesis of phenylmethyldiazomethane (PMDAM):
Acetofenone hydrazone: Acetofenone (2.0 g, 16 mmole) is diluted in 16 mL of absolute ethanol and then hydrazine (2.3 mL, 48 mmole) is added. It is carried back to the flow. After 2 h, the solvent is evaporated and the residue is taken back into ether (150 mL). Washed with water (100 mL). After drying on Na2SO4, the ether is evaporated. Pale yellow oil is obtained (1.5 g, 11 mmole, 69%). Phenylethylmethyldiazomethane (PMDAM): Hydrazone (150 mg, 1.1 mmole) is dissolved in 3 mL of THF. MnO2 (480 mg, 5.5 mmole) is added. It is stirred for 30 min at room temperature. The medium turns red. The solvent is filtered and evaporated. Red oil is obtained (145 mg, 100%) This reagent is used without purification.
Example 3.3: Synthesis of diphenyldiazomethane (DPDAM) is given below:
Benzophenone hydrazone is commercially available (reference B 960-2 Aldrich, France). The resulting 196 mg (1.0 mmole) is dissolved in 5 mL of THF. 435 mg (5 eq, 5.0 mmole) of MnO2 is added, the mixture is stirred for 10 minutes and then filtered. The recovered filtrate is evaporated dry.
Example 3.4 : Summary of the NVDAM:
The synthesis is carried out according to the protocol described above from 6-nitroveraatraldehyde (Aldrich, reference 27.960-9).
Example 4 : Synthesis of the biotinyl derivative from NPDAM:
The 2-amino-4-nitro benzaldehyde derivative is prepared by the method of ME Wall et al.
The preparation of NPDAM diazomethane is identical to that described in example 3.1 above.
Example 5 - Preparation of DNA nucleic acids
DNA amplifiers are generated by PCR from Mycobacterium tuberculosis 16S genomic DNA targets (10+4 copies as starting targets) using the Roche Fast Start Kit, 0.2 mM of each deoxyribonucleotide (d-ATP, d-CTP, d-GTP, d-TTP), 0.3 μM of primers and 0.4 μL of enzyme. The parameters of the PCR are as follows: The amplions are analysed qualitatively by electrophoresis on agarose gel (1.5%, TBE 0.5X). The deposited volume is 5 μL and the migration takes place for 20 min at 100 Volts (V). The PCR products are visualized under UV lamp after staining with ethidium bromide.
The conditions for culture, extraction of Mycobacteria and the start of amplification are given in patent application WO-A-99/65926.
Example 6: Reactivity of the labelling reagents on model nucleotides
The synthesis of the labelling reagents is described in examples 1 to 4 above. The PDAM described in the present invention is commercially available. (reference P1405, Molecular Probes, Eugene, OR)
Example 6.1 Marking of the UMP 3'-phosphate monomers
The reactivity of diazomethyl function labelling reagents has been studied to control reaction specificity.
A protocol has been developed to study this reactivity by capillary electrophoresis on a 3'-UMP model compound (Uridine 3'monophosphate, reference U1126 Sigma) under the following standard conditions: 3'-UMP 0,04 mM; H3BO3 2,0 mM; Marker 2,0 mM [added with an appropriate organic solvent (THF, AcOEt or DMSO) ]; Solvents H2O - CH3CN - Organic solvent (ratio: 1/3/1).
After centrifugation (5 min, 5000 rpm), the aqueous phase is analysed by capillary electrophoresis. The conditions of the capillary electrophoresis (EC) are as follows: the EC analysis was performed by the Beckman P/ACE 5000 apparatus. A capillary of non-melted silica (75 μm × 50 cm) was used. The voltage is applied at 30 kV (polarity) and the normal temperature of the capillary was maintained at 23 °C.The electrophoresis was recorded at 254 nm. The borate buffer solution (0.1 M, pH 8.3) was prepared from boric acid by adjusting the pH with a NaOH solution and filtered through a 0.2 μm filter. The samples were injected under pressure (0.5 psi, 5 seconds). Each analysis is preceded by capillary regeneration by successive passages of a NaOH (0.1 M, 2 min), water (2 min) and borate buffer (2 min) solution under pressure (20 psi).
The reaction is carried out by varying the reaction time between 0 and 4 hours as shown in each figure and the results are presented for each test reagent in Figures 2A to 2I with the concentration in reagent used. The reaction time is indicated on each electrophore chart. With all the reagents tested, the formation of a monoalkylated product is obtained, which proves the specificity of the reaction. The reactivity, i.e. the half-life of the reagent on the 3'-UMP, can be calculated by comparing the peak height and the results are given in Table 1 below (standard conditions described above): - What? Réactivité (temps de demi réaction) de réactifs
Formules chimiques et noms
Réactivité 5 min 20 h 30 min 4h
Formules chimiques et noms
Réactivité 10 h 15 min 5 min 1 min
Réactivité (temps de demi réaction) de réactifs
It should be noted, however, that since the reaction is very specific and does not lead to by-products for all test reagents, it is possible to increase the concentration of the labelling reagent without a consequence from the point of view of selectivity on the labelling. Thus, for the reagent DPDAM, if the concentration is increased to 20 mM (see Figure 2C), the reactivity (half-life) is 2 hours.
Example 6.2: Test of different 3'-monophosphate nucleotides:
To avoid any misinterpretation, a further study on the meta-marker BioPMDAM, taken as a significant example, was performed with the other 3'-monophosphate nucleotides.
The nucleotides tested are: 3'-AMP (reference 85,194-9, Aldrich), 3'-GMP (reference 151214, ICN), 3'-CMP (reference C1133, Sigma), 3'-TMP (deoxyribo series) (reference T1008, Sigma). The electrophoresis diagrams obtained with the different nucleotides are shown in Figures 3A to 3D. The reaction times shown in Figure 3A are the same for Figures 3B to 3D.
Regardless of the starting nucleotide (ribo or deoxyribo series), we observe the exclusive and complete formation of the alkylated product in 130 min at 60°C. It is important to note that in the case of guanine (the most reactive base with the usual alkylating reagents), only the alkylated product with phosphate is observed, proving the very high selectivity of the reaction. This study also allows to verify that the reaction rate does not depend on the nature of the nucleotide as a substrate.
Example 6.3: Study of a dinucleotide:
Alkylation of the dinucleotide ApUp (reference A4298, Sigma) was performed with meta-BioPMDAM to verify the selectivity of the reaction with respect to terminal phosphate versus internucleotide phosphate.
The formation of a single product is observed showing good selectivity of the meta-BioPMDAM reagent for terminal phosphate compared to internucleotide phosphate.
Example 6.4: Characterisation of adducts with the four (4) 3'-monophosphate nucleotides:
To ensure that the products obtained were indeed from phosphate alkylation, the adducts of the 3'-UMP, 3'-CMP, 3'-GMP and 3'-AMP monophosphates were synthesized with the reagent meta-BioPMDAM. The alkylation reaction is carried out at the preparatory scale as shown below. The adducts, obtained with yields of about 70%, are purified and then studied by proton and phosphorus NMR.
The preparation protocol:
The 3'-UMP (as disodium salt; 9.3 mg; 21.1 μmol) is dissolved in 2 mL of 0.1 M aqueous solution of H3BO3, then 2 mL of CH3CN, 6 mL of MeOH and then the reagent meta-BioPMDAM (75 mg; 0.20 mmol) are added successively. The reaction is carried out for 2.5 h at room temperature. It is followed by capillary electrophoresis. 3 mL of water is added, then the excess of the reagent is removed by extraction with CH2Cl2. The aqueous phase is evaporated. The residue is dissolved in a small amount of water and purified by passing over a column of reverse silica gel (Lichroproproproproprop RP-18, MeO; RP-18, MeO/OH2 (20/80);
The proton NMR spectra obtained for 3'-NMP adducts (N=G, U, C, A) are shown in Figures 5A to 5D. The identification of adducts was performed by two-dimensional 1H/1H (COSY) NMR experiments. Two diastereoisomers for each of these adducts in a 1/1 ratio are present. A single peak is present for the NMR of phosphorus at 0 ppm (300 MHz, D2O). These experiments show that the reaction is very specific, that only one adduct is observed and that the marking takes place on the phosphorus.
Example 7 - Study of temperature stability:
All diazomethane derivatives, as described in Table 1 of Example 6.1 above, shall be stored in the solid state in the freezer at -20 °C for at least three (3) months and no loss of reactivity shall be observed. The stability at room temperature on the mulch was determined by RMN 1H for both NPDAM and meta-BioPMDAM reagents. We observed no decomposition by leaving NPDAM on the mulch for one month without special precautions. We observed about 50% decomposition by leaving meta-BioPMDAM on the mulch for twenty-five (25) days. The temperature stability of the marking reagent is an essential characteristic.A reagent which is not stable for at least fifteen (15) days at -20°C, preferably one (1) month, is not marketable. Although there are means of storage and dispatch up to -110°C, there is a relationship between stability at -110°C and -20°C and therefore the value of fifteen (15) days at -20°C, preferably one (1) month at -20°C is an industrial minimum.And there's no easy way, like a carbon glass, to ship them from the manufacturer's point of view. As regards stability at room temperature, a stability of a few hours, preferably one (1) day, is sufficient to enable the user to perform the marking.
Example 8: Study of DNA fragmentation: Example 8.1 Hydrolysis of various nucleosides in acidic medium:
The aim of this study is to demonstrate the difference in acid pH stability between natural nucleosides, modified nucleosides and purine and pyrimidine nucleosides, and to improve control of DNA fragmentation by taking into account its base composition. - What?
Two modified nucleosides, 8-bromo-2'-deoxyadenosine (8-BrdA) and 5-bromo-2'-deoxycytidine (5-BrdC) as well as four (4) naturally occurring nucleosides (dA, dC, dG and dT) were used in this study. 50 nanomoles (nmol) of each nucleoside are incubated in 50 mM sodium formate pH 3 at 95°C. Incubation times range from 0 to 45 min. After vacuum drying and re-treating with 20 μl of pure water, samples (10 nmol) are then analysed by HPLC in reverse phase.
The curves in Figure 8 show that the modification of adenine at position 8 by a bromine atom makes this nucleoside less stable than the natural nucleoside. This study shows that DNA fragmentation by depuration or depyrimidination can be controlled by optimising hydrolysis conditions, or by incorporating either modified bases that are less stable than natural bases or bases that can be modified and hydrolysed after incorporation.
Example 8.2: Fragmentation of double stranded DNA incorporating or not a modified nucleotide:
Three PCR amplifications were performed in parallel from Mycobacterium tuberculosis 16S genomic DNA target (10+4 starting copies) using the Roche Fast Start Kit, 0.2 mM of each deoxyribonucleotide (d-ATP, d-CTP, d-GTP, d-TTP), 0.3 μM of primers and 0.4 μL of enzyme. The parameters of the PCR are those of example 5. In the first case, the protocol is used as is: the case of so-called natural PCR. In the second case, the protocol is modified to obtain a 30% PCR of 8Br-dATP. This is achieved by introducing 0.2 mM of d-CTP, d-GTP and d-TTP. 0.14 mM of d-ATP and 0.06 mM of 8-BrdATP are also introduced.
In the third case, the protocol is modified to obtain a 50% PCR of 8-BrdATP. This is achieved by introducing 0.2 mM of d-CTP, d-GTP and d-TTP.
The study of the fragmentation of these amplicons alone was carried out under the conditions described above: 50 mM sodium formate pH 3 at 95°C. The analysis was performed on polyacrylamide denaturing gel (8% polyacrylamide, 7 M urea, 1 X TBE) using ethidium bromide staining.
After 15 min incubation at 95 °C in 50 mM sodium formate pH 3, no difference was seen between the three (3) targets.
The PCR amplicon was also purified at different pH and at different temperatures and incubation times. Analysis on gel under the above conditions shows that at pH 3, fragmentation is total after only 10 min incubation at 95°C. At this pH, fragmentation is also total at 60°C after 30 min incubation. At pH 4, 30 min incubation is required to achieve complete fragmentation of DNA amplicons even at 95°C. This result is very interesting and shows that the base site generated at acid pH is unstable and therefore leads to fragmentation of the DNA strand without any other special treatment.
Example 9 DNA marking and fragmentation by the meta-bioPMDAM derivative (3a) in two steps:
The meta-bioPMDAM derivative (3a) was obtained according to the reaction scheme described in example 1.1. The DNA amplicons were prepared by PCR amplification according to the protocol described in example 5.
Marking
A 10 μl PCR, 38 μL pure water (sigma), 50 μL sodium formate at pH 3 (100 mM in pure water) is added and the mixture is incubated for 30 minutes at 60°C. Then 2 μL meta-bioPMDAM (100 mM in DMSO) is added next. The solution was vortex mixed and then incubated for an additional 15 minutes at 60°C. The tests shall be performed in duplicate to analyse DNA fragmentation on gel and the efficiency of DNA chip hybridisation and reading.
Cleansing
The purification is carried out on QIAQUICKTM columns (Nucleotide Removal Kit, Qiagen, Hilden, Germany) using the purification protocol recommended by the supplier.
After purification, the eluate is transferred to a clean tube containing 400 μL of hybridization buffer (1.75 mL 20X SSPE; 2.9 mL Betaine 5M; 290 μL DTAB 0.1M; 10 μL Antimouse 30%) are added. The reference beta is B-2754 Sigma, and the DTAB reference D-5047 Sigma. The solution is vortex mixed and incubated for 10 min at 95°C, to separate the DNA strands that are not separated during the marking step during the fragmentation (denaturation step).
The Commission shall adopt implementing acts laying down the rules for the application of this Regulation.
After the tagging step during fragmentation, the resulting fragments are hybridized on DNA chips designed for analysis of region 213-415 of the M20940 Genbank sequence of Mycobacterium tuberculosis 16S RNA. This DNA chip is described in A. Troesch et al, J. Clin Microbiol., 371), p49-55, 1999. The hybridization steps were performed at fluid stations (Affymetrix, Santa Clara, CA) using the hybridization protocol and buffers described in A. Troesch et al, J. Clin Microbiol., 37(1), p49-55, 1999.
Hybridisation is revealed by the coupling of streptavidine labelled with phycoerythrin (PE) which interacts with biotin in meta-BioPMDAM under the following conditions: 300 μL of pure water; 300 μL of Tris buffer 100 mM pH 7 / NaCl 1M / tween 0.05% /Antimouse 0.005%; 6 μL of BSA (50 mg/mL) ; 6 μL of streptavidine-PE (300 μg/mL). The following information is provided for the purpose of the analysis: Streptavidin-Phycoerythrin: reference R0438, Dako, Denmark,Streptavidin-CY5: reference C0050, Dako, Denmark,Antimousse reference M5-575, Ultra Additives Inc., andTween reference P-7949, Sigma.
DNA chip reading:
The reading of the fluorescence emitted on the surface of the DNA chip after marking and hybridization and the generation of data in terms of signal strength and percentage of homology are performed by the reading systems and software provided by Affymetrix (GeneChip® Instrument System and GeneChip® Information System, Santa Clara CA). The reading system provides signal and background noise intensities expressed in rfu ( relative fluorescence unit ). The percentage of homology is given with respect to a reference sequence which in this case is the sequence of Mycobacterium tuberculosis. The results in terms of average signal strength (I),the background noise (B) and the percentage of homology (%) are given in Table 2 below: Generally, a homology percentage greater than 90% is considered a satisfactory result although a result greater than 95% is generally sought. Values above 95% are no longer indicated as they are not significant in the case of the Mycobacterium DNA chip. A high intensity with low background noise is the second result sought in the following examples. In all results, background noise B is deduced from the mean intensity I.
Analysis on polyacrylamide gel
The samples to be analysed on gel shall be vacuum dried, with 10 μL of pure water and 10 μL of formamide 2X blue. The migration is carried out on 8% acrylamide gel in 1X TBE for one (1) hour at 150 V.
Acid pH has been used for DNA fragmentation, where the depuration phenomenon produces very unstable basic sites leading to almost immediate fragmentation of DNA sequences at high temperature, which produces DNA-5' phosphate fragments.
The gel analysis shows that incubation of PCR amplicons at 60°C for 30 min in solution in the formate buffer (50 mM, pH 3) leads to total fragmentation of these amplicons.
The results of the DNA amplitude fragmentation marking in terms of percentage homology, signal strength and background noise are given in Table 2 below. - What? Marquage et fragmentation des amplicons ADN en termes d'homologie, d'intensité des signaux (I) et du bruit de fond (B)
Tampon : formiate pH3, 50 mM
> 95 4456 593 7,5
Incubation : 30 min à 60°C
This example shows that the derivatives of the invention can be used for the marking of DNA fragments produced by enzymatic amplification in a two-step protocol. They can also be used for the marking of natural unamplified DNA.
Example 10 DNA marking by the derivative Cy5-PMDAM (12bis):
DNA marking by this new marker with diazomethyl function was assessed using a synthetic DNA fragment. - What?
The labelling reagent Cy5-PMDAM (12bis) shall be prepared according to the protocol described in Example 2. A twenty (20) mer oligodeoxyribonucleotide (ODN) is prepared using the phosphoramidite method. A phosphate is introduced at the 5' end by a standard phosphorylation reagent compatible with phosphoramidite chemistry. The sequence of this ODN consists of all natural DNA bases (ODN sequence: 5'-CTGAACGGTAGCATCTTGAC-3'). This sequence is complementary to the sequence of capture sequences of a DNA chip called model , synthesized using Affrixymetry technology.
Marking: 10 μL of Cy5-PMDAM (100 mM in DMSO) is added to 50 picomoles (pmoles) of this ODN. The final volume is 100 μL. After homogenization, incubation is carried out at 60°C for 30 minutes.
Purification and reading: Purification to remove excess marking reagent is performed as in Example 9.
Results:
The mean of the mark intensities (I) read on the DNA chip is 16 644 rfu for background noise (B) of 450 rfu. This level of intensity is very high and shows that the Cy5-PMDAM (12bis) marking reagent is fully compatible with the marking of DNA fragments on the phosphate group.
Example 11 DNA marking and fragmentation by PDAM reagent The following is the list of substances that are to be used for the labelling of the product:
PDAM is obtained from Molecular Probes (Eugene, OR) and solubilised in anhydrous DMSO. Two twenty (20) mer ODNs were used as DNA models: one 20mer 5'-hydroxyl ODN and the same 20mer ODN carrying a phosphate at the 5th end. The sequence of the ODN is described in Example 10. The marking reaction is carried out in a 50% DMSO and 1.5 mM 1-pyrenylidiazomethane (PDAM) mixture at 60°C for 30 minutes or one hour. The effectiveness of the marking was assessed by thin-layer chromatography (in normal phase) in an isopropanol/ammonia/water 60/10/30. After 30 minutes, the coupling is complete on the 5'-phosphate ODN. It takes one hour to obtain a partial coupling on the 5'-hydroxyl ODN i.e. about 50%.
The results of example 6 are confirmed on a 20-base model sequence for preferential labelling of reagents with diazomethyl function on the terminal phosphate. Labelling on an intranucleotide phosphate is not a redundant disadvantage as it may lead to increased sensitivity by introduction of more than one marker on the nucleic acid fragment. This allows the nucleic acid to hybridise with good sensitivity on the complementary target while maintaining good hybridisation specificity. The user can thus control the specificity of the marking by means of optimization reactions, for example by playing on the marking reagent, reaction time and temperature, to have a unique marking on the terminal phosphate.
Example 11.2: Kinetic study of the marking reaction by the PDAM:
This study was carried out using ODN 20mer 5'-phosphate under the previous conditions by varying the reaction time. The reverse phase column Spheri-5 RP-18 5 μm, 220 x 4.6 mm (Perkin Elmer). Buffer A: 0.1 M TEAA; Buffer B = 50% Buffer A + 50% CH3CN, and gradient of 10 to 50% B in 30 min to 1 ml/min at room temperature.
The results are shown in Figure 9 with the reaction time in minutes and the percentage of marking in order in abscissa. The yield is close to 90% after just 10 minutes of incubation at 60°C.
Example 11.3: Effect of temperature on the marking in the PDAM:
The labelling was carried out using ODN 20mer 5'-phosphate under the previous conditions varying the incubation temperature and with an incubation time of 10 minutes in each case.
The labelling efficiencies were assessed by reverse phase HPLC analysis. The results are shown in Figure 10 with the percentage of marking in order and the reaction temperature in °C in abscissa. It is very important to note that even at room temperature (25°C), we observe an ODN marking. After 10 minutes of incubation at 25°C, the marking efficiency is about 25%. At temperatures above 50°C, yields of over 80% are obtained. This shows the efficiency and flexibility of this DNA marking chemistry by reagents with a diazomethyl function.
Example 12 : Marking and fragmentation of DNA amplicons obtained by PCR amplification with the marking reagent meta-bioPMDAM (3a):
The meta-bioPMDAM derivative (3a) was obtained according to the reaction scheme described in example 1.1. The DNA amplicons were prepared by PCR amplification according to the protocol described in example 5.
Example 12.1: Comparison of marking with and without fragmentation: a. Marking under fragmentation conditions:
Add 50 μL sodium formate pH 3 (50 mM) and 2 μL meta-BioPMDAM 100 mM in DMSO to 10 μL of PCR. Adjust the volume to 100 μL. Incubate the solution for 30 min at 60°C.
b. Marking without fragmentation:
Add 2 μL of meta-BioPMDAM (100 mM in DMSO) to 10 μL of PCR. Adjust the volume to 100 μL. Incubate the solution for 30 min at 60°C.
The remainder of the protocol is identical to that in Example 9.
Results
Comparaison du marquage avec et sans fragmentation
>95 3995 569 7,0
94,1 500 542 0,9
The results in Table 3 above show that without fragmentation the average intensities obtained are at the same level as background noise (500 rfu). The marking during fragmentation gives a much higher intensity level (about 4000 rfu) and a very good percentage of homology.
Example 12.2: Effect of denaturation before DNA chip hybridisation:
Two labelling reactions were performed in parallel in two separate tubes according to the following protocol: 50 μL of sodium formate buffer pH 3 (50 mM) and 2 μL of meta-bioPMDAM (100 mM in DMSO) are added to 10 μL of PCR, the total volume is adjusted to 100 μL and incubated for 30 min at 60°C. After column purification (example 9), the solution from the first tube is incubated for 10 min at 95°C (to remove the double strand of DNA) and then the tube is immersed in an ice-water mixture at 0°C until hybridised on the DNA chip. The solution from the second tube is hybridised on the DNA chip without prior denaturation.
The hybridized biotinylated fragments on the capture probes on the DNA chip surface are detected by introducing a phycoerythrin-labeled streptavidine using the conditions described in Example 9.
Results
Effet de la dénaturation avant hybridation sur puce à ADN
>95 22812 570 40,1
93,5 4795 681 7,0
The results obtained, shown in Table 4, with pre-hybridation denaturation are superior to those obtained without the denaturation step. This shows that DNA denaturation is necessary to obtain a good level of intensity. Fragmentation via basic sites is one way to facilitate the denaturation of a double stranded DNA and strengthen hybridation on capture probes. To test other labelling reagents and taking into account the results obtained under the various conditions above, we defined a reference protocol using sodium formate buffer fragmentation (50 mM, pH 3) and a pre-hybridation denaturation step.
Example 13 : Marking and fragmentation of PCR amplicons by biotinised reagents in a one-step protocol:
The meta-, ortho- and para-bioPMDAM derivatives were prepared according to the protocol described in examples 1.1, 1.2 and 1.3. They were solubilized in anhydrous DMSO at a concentration of 100 mM.
The protocol is identical to that of example 12.2 above (marking and fragmentation in one step and then denaturation step pre-hybridation).
Results
Marquage et fragmentation des amplicons PCR par les réactifs biotinylés dans un protocole en une étape
>95 25951 820 31,6
>95 22960 581 39,5
94,1 43785 1205 36,3
The optimised protocol with one-step fragmentation and marking gives excellent results with different marking reagents with diazomethyl reactive function, as shown by the results in Table 5.
Example 14 DNA marking and fragmentation by BioDPDAM
The DNA amplicons were prepared by PCR amplification according to the protocol described in example 5. The synthesis of the labelling reagent is described in Example 1. The protocol is identical to that in Example 12.2, including denaturation at 95 °C before the hybridisation step.
Results
Marquage et fragmentation de l'ADN par le BioDPDAM
BioDPDAM 93,0 32359 3610 9,1
This result, as described in Table 6, shows that substitutions as large as the phenyl group can be used to optimise the reactivity of diazomethyl function labelling reagents.
Example 15 DNA marking and fragmentation by 5-bromomethylfluorescein
The DNA amplicons were prepared by PCR amplification according to the protocol described in example 5. Add 50 μL sodium formate pH3 (100 mM) and 2 μL 5-bromomethyl) fluorescein (Molecular probes, Eugen, OR) (100 mM in DMSO) to 10 μL of PCR. Adjust the volume to 100 μL. Incubate the solution for 30 min at 60°C.
The purification conditions are as shown in Figure 9. A denaturation step is carried out as shown in Figure 12.2. The other hybridization and reading conditions are identical to those described in the article by A. Troesch et al. J. Clin. Microbiology, 37 ((1), p. 49-55 1999. Marquage et fragmentation de l'ADN par la 5-(bromométhyl)fluorescéine
>95 855 183 4,7
This result from Table 7 shows that DNA fragmentation by the creation of basilic sites is fully compatible with a marking reagent with an alkyl halogenide reactive function.
Example 16: Marking and fragmentation of DNA amplicons in the presence of another chemical fragmentation agent derived from phenanthroline:
DNA amplifiers were prepared by PCR amplification according to the protocol described in Example 5. Two types of conditions are used:
Conditions a) are:
Add 20 μL of phenanthroline-FeSO4 (25 mM) and 2 μL of meta-BioPMDAM (100 mM in DMSO) to 10 μL of PCR. Adjust the total volume to 100 μL. Incubate the mixture for 60 min at 95°C.
Conditions b) The following:
Add 50 μL of sodium formate buffer pH3 (100 mM in pure water) and 2 μL of meta-BioPMDAM (100 mM in DMSO) to 10 μL of PCR. Adjust the total volume to 100 μL. Incubate the mixture for 60 min at 95°C.
The other conditions of the protocol are identical to those in Example 9. Marquage et fragmentation des amplicons ADN en présence de la phénanthroline
>95 2236 500 4,5
>95 6786 565 12,0
Both fragmentation conditions allow a satisfactory result as shown in Table 8. The best result is obtained with conditions (b) using acid pH fragmentation.
Example 17: Fragmentation of PCR amplicons marked by incorporation of d-UTP-fluorescein: Incorporation of the marked nucleotide
A PCR amplification was performed according to the following protocol to generate fluorescein-marked PCR amplifiers (baseline marking). From Mycobacterium tuberculosis 16S genomic DNA target (10+4 copies) using Roche's Fast Start kit, 0.2 mM of ATP, CTP and GTP deoxyribonucleotides as well as 0.14 mM of TTP and 0.06 mM of UTP-12-fluorescein, 0.3 μM of amorces and 0.4 μL of enzyme. The percentage of the marked nucleotide relative to its natural d-UTP counterpart is 30%. This ratio is generally used in the amplicotic marking reactions by incorporation of marked nucleotides. d-UTP-12-fluorescein is commercially available from Roche Diagnostics reference 1373242, Mannheim, Germany). The parameters of the PCR are those of example 5.
a. Fragmentation of PCR amplicons marked at 30% by d-UTP-fluorescein:
Add 50 μL of sodium formate buffer pH 3 (50 mM) to 10 μL of PCR. Adjust the volume to 100 μL. Incubate the solution for 30 min at 60°C.
b. Marking during the fragmentation of PCR amplicons containing 30% d-UTP-fluorescein:
A 10 μL PCR is followed by 50 μL sodium formate buffer pH 3 (50 mM) and 2 μL meta-BioPMDAM (100 mM in DMSO) and the volume is adjusted to 100 μL. The solution is then incubated for 30 min at 60°C. This test is a reference protocol that allows the comparison of different labelling strategies without the variability due to the amplification step.
A column purification step and a 95°C denaturation step are performed in all cases as in example 9.
The following shall be added:
Nucleic acids obtained by fragmentation of amplicons labelled with d-UTP-fluorescein (conditions a)) are hybridised on a DNA chip and first detected by direct reading of fluorescent signals emitted by fluorescein as described in Example 15.
The following shall be added:
A signal amplification step was used to improve the labelling sensitivity. The signal amplification was achieved by introducing a biotinyled anti-fluorescein antibody (reference 216-065-084, Jackson ImmunoResearch) and then a phycoerythrin-labelled streptavidine during the hybridisation step using the following successive conditions: 300 μL of pure water,300 μL of Tris 100 mM pH 7 / NaCl 1M / tween 0.05% / Antimouse 0.005% ,2.4 μL of BSA (50 mg/ mL),1.2 μL of biotinyl anti-fluorescein antibodies. (1 mg/ mL),300 μL of pure water, and300 μL of Tris 100 mM pH 7 / NaCl 1M / tween 0.05% / Antimouse 0.005% 6 μL of BSA; (50 mg/ mL) 6 μL of streptavidine-PE. (300 μg/ mL). In this protocol, fluorescein acts as a hapten (a tracer detectable indirectly by a marked antibody) and not as a fluorophore.
Protocol (b) is amended as follows:
Biotinyled (condition b) DNA chip hybrid fragments are detected by introduction of a phycoerythrin-labelled streptavidine using the following conditions: 300 μL of pure water, and 300 μL of Tris buffer 100 mM pH 7 / NaCl 1M / tween 0.05% / Antimouse 0.005%; 6 μL of BSA (50 mg/ mL) ; 6 μL of streptavidine which is labeled (300 μg/ mL).
The reading of the fluorescence emitted on the surface of the DNA chip after marking and hybridization and the generation of data in terms of signal strength and percentage of homology are carried out by the reading systems and software provided by Affymetrix. Fluorescein in case the ampoules are marked with the UTP-fluorescein only, according to Protocol Al, or phycoerythrin in case the ampoules are marked: The d-UTP-fluorescein with signal amplification, according to protocol a2, or the meta-bioPMDAM during their fragmentation, according to protocol b. In both cases where the detection is by phycoerythrin, the use of a filter allows the signal generated by fluorescein to be released and the phycoerythrin signal is detected.
Results
Fragmentation des amplicons PCR marqués par incorporation de d-UTP-fluorescéine
Flu* 81,6 595 342 1,7
PE* >95 22107 3461 6,4
PE* >95 21700 1503 14,4
Fragmentation des amplicons PCR marqués par incorporation de d-UTP-fluorescéine
* Flu =Fluorescéine et PE = Phycoérythrine
The results in Table 9 above show that chemical fragmentation using basilic site creation is compatible with enzymatic marking of DNA amplicons and that marking can take place prior to fragmentation. The intensity levels and percentage of homology obtained with this enzyme fluorophore incorporation protocol are low compared to those obtained with the marking during the fragmentation using the marking reagent with a diazomethyl function such as meta-bioPMDAM (conditions (b)). To reach the intensity level obtained with the meta-bioPMDAM derivative, a signal amplification step is required (conditions a2). This shows the effectiveness of the diazomethyl reactive function compared to the traditional modified basic incorporation such as fluorescein d-UTP (reference protocol (b)).
Example 18 : Fragmentation of double stranded DNA by sonication:
DNA amplicons were obtained using the protocol described in Example 5. These amplicons were fragmented by sonication in the presence and absence of the marker.
a. Marking of PCR amplifiers during sonication:
The mixture is incubated for 30 min at 60°C in an ultrasonic bath (frequency 35kHz, model T460-H, Bioblock, France).
The following shall be used for the calculation of the concentration of the test chemical:
Add 50 μL sodium formate pH 3 (50 mM) and 2 μL meta-BioPMDAM (100 mM in DMSO) to 10 μL of PCR reaction and adjust the volume to 100 μL. Incubate the solution for 30 min at 60°C.
The tests shall be performed in duplicate to analyse DNA fragmentation on gel and the efficiency of DNA chip hybridisation and reading as described above (Example 9 on phycoerythrin detection).
Analysis on the gel
The analysis was performed on polyacrylamide denaturing gel (8% polyacrylamide, 7 M urea, 1 X TBE) using ethidium bromide staining. The gel analysis shows that the DNA amplicons are fragmented by sonication at 60 degrees.
Results
Fragmentation de l'ADN double brin par sonication
93,8 2271 631 3,6
>95 19639 1459 13,5
The results of the marking in Table 10 during sonication (conditions a) are satisfactory, showing that the physical fragmentation of DNA targets by sonication is compatible with the marking chemistry by marking reagents with a diazomethyl function. The low marking results in this case are certainly due to the fact that the marker degrades under the effect of ultrasound.
Example 19 - Marking, fragmentation and denaturing of DNA in one step
The DNA amplicons were prepared by PCR amplification according to the protocol described in example 5. Two marking reactions were performed:
a. Marking, fragmentation and denaturing at 95 °C in one step:
To 10 μL of PCR add 50 μL of sodium formate pH3 buffer (50 mM) and 2 μL of meta-BioPMDAM marker (100 mM in anhydrous DMSO). The final volume is adjusted to 100 μL. The solution is then incubated for 30 min at 95°C. In this case, the reaction mixture was hybridized on a DNA chip without any prior purification.
b. Marking and fragmentation at 60 °C:
A 10 μL PCR is followed by 50 μL of sodium formate pH3 buffer (50 mM) and 2 μL of meta-BioDPDAM (100 mM in anhydrous DMSO). The final volume is adjusted to 100 μL. The solution is then incubated for 30 min at 60°C. The reaction mixture was then purified according to the protocol described above. In this protocol and prior to DNA chip hybridization, the fragments were denatured according to the protocol described in example 12.2.
Results
Marquage, fragmentation et dénaturation de l'ADN en une étape
>95 5426 506 10,7
>95 7015 818 6,8
These results in Table 11 show that with the basal site-building fragmentation approach, DNA marking, fragmentation and denaturation can be accomplished in a single step, which represents a marked improvement in terms of simplicity and time for the user without affecting sensitivity.
The following is the list of active substances:
• Protection of 4-carboxylbenzaldehyde:
4-carboxybenzaldehyde is commercially available. It is dissolved (3 g; 20 mmol) in a solution of trimethylsilyl chloride (10 g; 92 mmol) in 100 mL of MeOH. The mixture is left to agitate for 40 h at room temperature. After evaporation, a white solid corresponding to 4-methoxycarbonylbenzaldehyde 24 is isolated, characterized by NMR and used as such for the next reaction.
The measurement of the concentration of the test chemical is based on the following equation:
•Protection of 4'-methoxycarbonylbenzaldehyde:
The 4-methoxycarbonylbenzaldehyde (3.35 g; 20 mmol) is dissolved with trimethyl orthoformiate (4.8 g; 40 mmol) in the presence of Dowex 50WX8-100 (1 g). The mixture is heated at reflux for 2 h, then filtered and evaporated. After a recrystallization test, an MRI analysis shows that the reaction is not complete and the reaction is resumed in 30 mL of MeOH, 30 mL of CHOMe3 and 1 g of Dowex 50WX8-100 at room temperature. Filtration and evaporation are performed, to obtain 3.55 (16.89 gol, 84 mmol) of product 25.
The measurement of the concentration of the test chemical is based on the following equations:
•Compound 26:
The solution obtained is heated to 140-150°C for 2 h. The mixture is then dissolved in 100 mL of DCM (dichloromethane or CH2Cl2) and washed 6 times with 10 mL of water. The organic phase is dried with evaporated Mg4 SO until an oil is obtained. This oil is then washed with pentane 3 times in a row by decantation, then further extraction with DCM and H2O is performed. After drying on Mg4SO and evaporation, the product 26 is isolated with an evaporation of 63% (9,27 mmol).
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• Biotinyl compound 27:
The biotin (500 mg 2.05 mmol) is suspended in 10 mL DMF and then 365 mg (2.25 mmol) CDI is added. This solution is left to agitate for 30 min at room temperature. Compound 26 (900 mg; 2.26 mmol) is dissolved in 1 mL DMF and then added gradually to the previous solution. The resulting mixture is left to agitate for 1 h at room temperature. After evaporation, a column purification by flash chromatography (column 20 mm diameter) is performed with 250 mL MeOH-DCM 6%, then with 200 mL MeOH-DCM 7%, and finally with 200 mL MeOH-DCM 8%. The fractions corresponding to the 27 g product are then evaporated to give an estimated oil yield of 1.00%.
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•Aldehyde compound 28:
The organic phase is recovered and dried on anhydrous NaHCO3. The compound 28 is filtered, evaporated and obtained as a paste (495 mg; 0.855 mmol) with an overall yield of 42% from biotin.
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• Compound hydrazone 29:
The aldehyde 28 (495 mg; 0.855 mmol) is dissolved in 10 mL of absolute ethanol. Hydrazine (350 μL; 7.20 mmol) is added and the reaction mixture is then heated at low temperature for 1 h. The oil obtained after evaporation is dissolved in EtOH abs, to be re-evaporated. A foam is then obtained and crushed in pentane. The paste corresponding to product 29 (511 mg; 0.862 mmol) is obtained with a yield of 100%.
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• Diazo compound 30:
Hydrazone 29 (357 mg; 0.602 mmol) is dissolved in 17.5 mL DMF. MnO2 (700 mg; 7.7 mmol) is then added. After 12 min of stirring at room temperature, the mixture is filtered on a millipore containing cellulite (thickness: 2 cm) and molecular sieve into a powdered 3 Å (0.5 cm). The reaction mixture is evaporated dry. The resulting residual oil is washed with ether three times in a row. Compound 30 (290 mg; 0.491 mmol) is obtained as a slightly pink solid with an efficiency of 82%.
The following are the main components of the test method: the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the test medium, the measurement of the test medium, the measurement of the test medium, the measurement of the test medium, the measurement of the test medium, the measurement of the test medium, the measurement of the test medium, the measurement of the test medium, the measurement of the test medium, the measurement of the test medium, the test method of the test medium, the measurement of the test medium, the test, the measurement of the test, the measurement of the test, the measurement of the test, the measurement of the test, the test, the measurement of the test, the test, the measurement, the measurement, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test,
The reactivity of compound 30 was tested on 3'-Urdine monophosphate and followed by capillary electrophoresis. The reagent is stable at -20 °C for at least 1 month.
Example 21: Marking and fragmentation of DNA amplifiers with the para-Bio-EG3-PDAM marking reagent:
The main interests of these types of molecules, i.e. PDAM derivatives with a polyethylene glycol-based binding arm, are to allow the diazo function to be removed from biotin and to increase the solubility and ultimately reactivity of these molecules.
The para-Bio-EG3-PDAM 30 derivative was obtained according to the reaction pattern described in Example 20. The DNA amplifiers were prepared by PCR amplification according to the protocol described in Example 5.
a. Labelling by the reagent para-Bio-EG3-PDAM:
10 μL of PCR is followed by 10 μL of para-Bio-EG3-PDAM (100 mM in DMSO) and 77 μL of Dnase/Rnase free water. The solution is homogeneous and has no precipitation. This solution is incubated for 10 min at 95°C, then 3 μL of HCl at 0.1 M is added and the solution is incubated for 10 min at 95°C.
The remainder of the protocol is identical to that in Example 8.
b. Marking by the reagent meta-BioPMDAM:
10 μL of PCR is made up of 10 μL of meta-BioPMDAM (100 mM in DMSO) and 77 μL of Dnase/Rnase free water. The synthesis of this product is described in example 1.1. The remainder of the protocol is identical to that in Example 8.
Results:
Etude comparative du marquage et de la fragmentation d'amplicons d'ADN avec le -Bio-EG3-PDAM et le -BioPMDAM
> 95% 15151 621 24,4
> 95% 11226 515 21,8
The signal intensities obtained in Table 12 are very satisfactory and the homology percentage is high. This result shows that the introduction of a polyethylene glycol arm on the diazo-marking molecule increases the solubility in the aqueous phase of the reagent. The test is therefore homogeneous.
Example 22: Synthesis of other PDAM derivatives with a polyethylene glycol-based bonding arm: The following is a summary of the results of the analysis: The following table summarizes the results of the analysis:
Compound 68:
The 3-aminoacetophenone (14.5 g, 107 mmol) is dissolved in 50 ml of anhydrous DMF. Succinic anhydride (10.7 g, 107 mmol) is added and left to agitate, argon and room temperature. After 6 h, the solution is concentrated under vacuum and 50 ml of methanol is added. The precipitate is filtered and washed with methanol and ether. The frequency range of the measurement is defined as the frequency range of the measurement.
Compound 69:
After 20 min, 20 ml (94.6 mmol) of 4, 7, 10-trioxatridecanediamine (EG3) is slowly added.[3] After 3 hours of reaction at room temperature, the DMF is evaporated and the residue is taken up again in 100 ml of CH22. Extractions are made with saturated NaHCO3 and H2O, after which the organic phase is dried with Na2SO4 hydrate and the evaporated solvent. 4.34 g (46%) of the product is obtained.
Biotinyl compound 70:
D-biotin (1.0 g, 4.1 mmol) is dissolved in 10 ml of anhydrous DMF under argon. It is cooled on ice and carbonyldiimidazole (CDI) (0.665 g, 4.1 mmol) is added to 10 ml of anhydrous DMF. After 15 min, compound 69 (1.8 g, 4.1 mmol) is added to 2 ml of anhydrous DMF. It is allowed to react for 3 h at 35°C, then DMF is evaporated and taken up again in 100 ml of CHCL22. Extractions are made with saturated NaHCO3 and H2O, after which the organic phase is dried with anhydrous Na2SO4 and soil evaporates. The final compound, meta-Bio-EG3-PMDAM, is obtained after two synthesis steps as shown in Example 1.
The interest of this synthesis is twofold: on the one hand, the product 69 is obtained in only two steps; this product can be used as a precursor to diazo with the possibility of attaching detectable molecules of different nature to it, thanks to the terminal amine grouping; this grouping also allows the grafting of the compound 69 on solid media, with the aim of immobilizing nucleic acids; on the other hand, the compound 71 has the same reactive center as the meta-Bio-PMDAM (re-designated reference molecule), which facilitates the analysis of the advantages associated with the inclusion of the ethylglycol arm (EG3). The reagent is stable at -20 °C for at least 1 month.
The following is the list of active substances: The following is a summary diagram:
• Protection of 3-nitroacetophenone 13:
33 g (0.20 mol) of 3-nitroacetophenone is dissolved in 400 mL of toluene, then 40 mL (0.717 mol) of ethylene glycol and 600 mg (3.15 mmol) of para-toluene sulfonic acid (APTS) are added. A Dean Stark system is mounted. The solution is heated for 3 hours at 130°C. After allowing the solution to return to room temperature, 400 mL of ethyl acetate is added, then washed with 8 mL of a saturated NaHCO3 solution. The organic phase is dried on MgSO4.
The mean of the measurements is calculated by multiplying the mean of the measurements by the mean of the measurements.
• Preparation of amine 14
Compound 13 (39.7 g; 0.190 mol) is dissolved in 500 mL of EtOH, then 1 g of palladium on 10% charcoal is added. Heat to dissolve everything, then let the solution return to room temperature. After vacuuming and putting the solution under H2, let it be stirred for 5 h. The solution is then hot-filtered and then evaporated.
The mean value of the measurement is calculated as the sum of the values of the measurements of the two samples taken together.
• Compound bromide 15:
Amine 14 (12.3 g; 68.7 mmol) and triethylamine (7 g; 69 mmol) are dissolved in 150 mL of DCM under argon. A solution of 13.8 g (60 mmol) of bromoacetyl bromide dissolved in 150 mL of DCM is added at 5 °C by drip. At the end of the addition, 100 mL of NaHCO3 aqueous 1 N is added. The organic phase is washed twice in a row with NaHCO3 aqueous and dried on MgSO4. After dry evaporation, 22.6 g of brown oil corresponding to compound 15 is obtained and used for the following reaction.
The following are the main components of the test method:
•Alcohol compound 16:
The sodium hydride (3.3 g; 82.5 mmol) is washed three times with pentane and then suspended in 150 mL of THF, ethylene tetra glycol (50 mL; 0.29 mol) is then added at room temperature. The solution is concentrated at 100 mL, then diluted with 500 mL of CHCl3. This organic phase is washed three times with 250 mL of NaHCO3 aqueous 1 N, then dried on MgSO4 before evaporation. The product is purified on a silica column by flash chromatography (column 65 mm diameter) with 1.5 L of Me-OHDCM 5%, then with 500 mL of Me-OHDCM 7%, and finally with 500 mL of Me-OHDCM 10%. The fractions corresponding to the 16 percent compound are collected, then evaporated to give 17,4 g (12,1 mm) of 61% drying product.
The following equations are used for the determination of the concentration of the active substance in the feed additive:
• Toxylate 17 compound:
The alcohol 16 (4,13 g;10,0 mmol) is dissolved in 5 mL of pyridine. Then, 2.0 g (10,5 mmol) of tosyl chloride is added at room temperature. It is stirred under argon for 10 h. It is diluted with 100 mL of DCM, washed three times with 20 mL of NaHCO3 aqueous 1 N, then dried on MgSO4 before co-evaporating with toluene. A column purification by flash chromatography (column 50 mm diameter) is performed with 500 mL of MeOH-DCM 2%, then 500 mL of MeOH-DCM 3%, and finally 500 mL of MeOH-DCM 4%. The fractions corresponding to the 17 percent product are evaporated and then collected to yield 65 g (6.48 mmol) with an estimated oil yield of 3.48%.
The following are the main components of the test method: - the measurement of the concentration of the test substance in the test medium; - the measurement of the concentration of the test substance in the test medium; - the measurement of the concentration of the test substance in the test medium; - the measurement of the concentration of the test substance in the test medium.
• Phthalimide 18 is a compound:
The resulting solution is heated to 85-90°C for 17 h and then evaporated. The product is purified on a silica column by flash chromatography (column 50 mm diameter) with 1 L of acetone-DCM 15%, then with 1 L of acetone-DCM 20%. The fractions corresponding to compound 18 are collected and then evaporated dry to give 3.15 g (5.8 mmol) of product, with an efficiency of 90%.
The following are the main components of the test method:
•Amine compound 19:
The product 18 is dissolved in 20 mL of absolute EtOH by reflux heating at 75-80°C. Hydrazine (1.07 mL; 22.1 mmol) is then added, and the reaction is agitated for 1 h 15. The precipitate obtained is filtered on a fritted basis and the ethanol phase evaporated. The white precipitate is then washed in the DCM, and the DCM phase is evaporated. The yellow oil obtained (2.3 g; 5.57 mmol) is directly used for the next reaction, although it contains imidazole which can be removed later, during the acetal unprotection step.
The following equation is used for the determination of the concentration of the active substance in the feed additive:
• Biotinyled compound 20:
D-Biotin is solubilised (1.05 g; 4.32 mmol) in 10 mL of anhydrous DMF. 790 mg (4.87 mmol) of carbonyl diimidazole (CDI) is added under argon. After 10 min of agitation, amine 19 is added diluted in 5 mL of DMF. The solution is left to agitate for 40 min, then evaporated before being purified on column by flash chromatography. For this purpose, a 50 mm diameter column is used with 500 mL of MeOH-DCM 5%, then 500 mL of MeOH-DCM 10%, and finally 500 mL of MeOH-DCM 15%, as the electrolyte. The resulting yellow oil (2.4 g) contains about 30% by weight of imidazole according to the NMR spectrum, which implies that the yield of the reaction resulting in product 20 is about 60% compared to the starting biotin.
The mean of the measurements is calculated by dividing the mean of the measurements by the mean of the measurements by the mean of the measurements.
•Compound ketone 21:
Acetal 20 is dissolved in 80 mL of chloroform, then 30 mL of HCl 2 N is added. The organic phase is recovered and then dried on anhydrous NaHCO3. After filtration, the solution is evaporated and the oil obtained is washed with pentane to give the product 21 (1.48 g; 2.48 mmol) with an efficiency of 99%.
The following are the main components of the test method: the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the test medium, the measurement of the pH of the test medium, the measurement of the test medium, the measurement of the test medium, the measurement of the test medium, the measurement of the test medium, the measurement of the test medium, the measurement of the test medium, the measurement of the test medium, the measurement of the test medium, the measurement of the test medium, the test, the measurement of the test medium, the test, the measurement of the test, the measurement of the test, the measurement of the test, the test, the measurement of the test, the test, the measurement, the measurement, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the
• Compound hydrazone 22:
Ketone 21 is dissolved in 20 mL of absolute EtOH. It is heated at 75 -80°C. Hydrazine (816 μL; 16.81 mmol) is added and stirred for 3 h. After filtration, it is evaporated dry, dissolved in ethanol until a sticky white foam is obtained. In a second step, this foam is dissolved in 50 mL of chloroform and then 20 mL of a saturated NaHCO3 solution is added. It is washed well and then the organic phase is recovered. It is dried on anhydrous Na2CO3 and after filtration, it is evaporated dry to obtain a new sticky foam.
The following are the main components of the test method: the test method is based on the following equation:
• Diazo compound 23:
Hydrazone 22 (100 mg; 0.164 mmol) is dissolved in 1 mL of DMF under argon. 80 mg of activated MnO2 is added and stirred for 30 min. The mixture is filtered through a mixture of Cellite (3 cm) -molecular sieve powder (1 cm) layer. The solution is then evaporated dry. The oil obtained at the end of evaporation is crushed to a pink powder corresponding to compound 23 (78 mg; 0.128 mmol; 78 %).
The following are the main components of the test method: the measurement of the radiation emitted by the test vessel, the measurement of the radiation emitted by the test vessel, the measurement of the radiation emitted by the test vessel, the measurement of the radiation emitted by the test vessel, the measurement of the radiation emitted by the test vessel, the measurement of the radiation emitted by the test vessel, the measurement of the radiation emitted by the test vessel, the measurement of the radiation emitted by the test vessel, the measurement of the radiation emitted by the test vessel, the measurement of the radiation emitted by the test vessel, the measurement of the radiation emitted by the test vessel, the measurement of the radiation emitted by the test vessel, the measurement of the radiation emitted by the test vessel, the measurement of the radiation emitted by the test vessel, the measurement of the radiation emitted by the test vessel, the measurement of the radiation emitted by the test vessel, the measurement of the test vessel, the measurement of the radiation emitted by the test vessel, the measurement of the test vessel, the measurement of the test vessel, the measurement of the test vessel, the measurement of the test vessel, the measurement of the test vessel, the measurement of the test vessel, the measurement of the test vessel, the measurement of the test vessel, the measurement of the test vessel, the measurement of the test vessel, the measurement of the test vessel, the measurement of the test vessel, the measurement, the measurement of the test vessel, the measurement, the measurement, the measurement of the measurement, the measurement, the measurement, the measurement, the measurement, the measurement, the measurement, the measurement, the measurement, the measurement,
The reactivity of compound 23 was tested on 3'-Urdine monophosphate and followed by capillary electrophoresis. The stability of the reagent is more than 1 month at -20 °C.
The following is the list of active substances: The following is a summary diagram:
• Protection of 4-acetylbenzoic acid:
The mixture is heated to 90°C overnight. After evaporation, a white solid corresponding to compound 31 (1.21 g; 5.75 mmol) is isolated, characterised by NMR and used as such for the next reaction. The measurement of the concentration of the test chemical is based on the following equation:
• Composed of 32:
The mixture is heated to 60°C overnight, then filtered and evaporated to give the compound 32 (1.19 g; 5.3 mmol) with an efficiency of 87%. The measurement of the concentration of the test chemical is based on the following equations:
•Composite 33:
The solution obtained is heated to 140°C for 4 h. The mixture is then dissolved in 30 mL of DCM and washed 3 times with 10 mL of water. The organic phase is dried with Mg SO4 and then evaporated to an oil corresponding to product 33 (1.44 g; 3.49 mmol) with an efficiency of 67%.
The following are the main components of the test method:
• Biotinyled compound 34:
The biotin (780 mg 3.19 mmol) is suspended in 13 mL DMF. 590 mg (3.60 mmol) CDI is then added. This solution is left to agitate for 30 min at room temperature. Compound 33 is dissolved in 1 mL DMF and then added gradually to the previous solution. The resulting mixture is left to agitate for 1 h at room temperature. After evaporation of DMF, a column purification by flash chromatography (column 35 mm diameter) is performed with 500 mL MeOH-DCM 6%, then with 250 mL MeOH-DCM 8%, and finally with 250 mL MeOH-DCM 8%. The fractions corresponding to the 34 g of the product are collected and then evaporated to give an estimated dry oil yield of 30%.
The following are the main components of the test method: - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical in the test chemical, - the measurement of the concentration in the test chemical in the test chemical in the test chemical, - the measurement of the concentration in the test chemical in the test chemical in the test chemical in the test chemical, - the measurement of the concentration in the test chemical in the test chemical in the test chemical in the test chemical in the test chemical in the test chemical, - the measurement of the test chemical in the test chemical in the test chemical in the test chemical in the test chemical in the test chemical in the test chemical in the test chemical in the test chemical in the test chemical in the test, - the measurement in the test chemical in the test chemical in the test chemical in the test chemical in the test chemical in the test chemical in the test chemical in the test, - the test chemical in the test in the test chemical in the test chemical in the test in the
•Composite 35:
The acetal 34 is dissolved in 45 mL of chloroform, then 10 mL of HCl 2N is added. The diphasic mixture is vigorously agitated for 5 min. The organic phase is recovered and dried on anhydrous NaHCO3.
The following are the main components of the test method: the measurement of the concentration of the test substance in the test chemical, the measurement of the concentration of the test substance in the test chemical, the measurement of the concentration of the test substance in the test chemical, the measurement of the concentration of the test substance in the test chemical, the measurement of the concentration of the test substance in the test chemical, the measurement of the concentration of the test substance in the test chemical, the measurement of the concentration of the test substance in the test chemical, the measurement of the concentration of the test substance in the test chemical, the measurement of the concentration of the test substance in the test chemical, the measurement of the concentration of the test substance in the test chemical, the measurement of the concentration of the test substance in the test chemical, the measurement of the concentration of the test substance in the test chemical, the measurement of the concentration of the test substance in the test chemical, the measurement of the concentration of the test substance in the test chemical, the measurement of the concentration of the test substance in the test chemical, the measurement of the concentration of the test substance in the test chemical, the measurement of the concentration of the test substance in the test chemical, the measurement of the concentration of the test substance in the test chemical, the measurement of the test substance in the test chemical, the measurement of the concentration in the test substance in the test chemical, the measurement of the test substance in the test substance in the test chemical, the measurement of the test substance in the test substance in the test chemical, the measurement of the test substance in the test substance in the test chemical, the measurement of the test substance in the test substance in the test substance in the test substance, the measurement in the test substance in the test substance in the test substance, the test substance in the test substance in the test substance, the test substance in the test substance in the test substance, the test substance in the test substance in the test substance, the test substance in the test substance in the test substance, the test substance in the test substance, the test substance in the test substance in the test substance, the test substance in the test substance in the test substance, the test substance in the test substance, the test substance in the
• Compound hydrazone 36:
The ketone 35 (500 mg; 0.864 mmol) is dissolved in 11 mL of absolute EtOH. Hydrazine (335 μL; 6.911 mmol) is added, and then the reaction mixture is heated at reflux for 1 h. The oil obtained after evaporation is dissolved in EtOH abs, to be evaporated again.
The following are the main components of the test method: the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the pH of the test medium, the measurement of the test medium, the measurement of the pH of the test medium, the measurement of the test medium, the measurement of the test medium, the measurement of the test medium, the measurement of the test medium, the measurement of the test medium, the measurement of the test medium, the measurement of the test medium, the measurement of the test medium, the measurement of the test medium, the measurement of the test medium, the test, the measurement of the test medium, the test, the measurement of the test, the measurement of the test, the measurement of the test, the test, the measurement of the test, the test, the measurement, the measurement, the measurement, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test, the test
• Diazo compound 37:
The reaction mixture is evaporated dry. The resulting residual oil is washed with ether three times in succession until it is a powder. The compound 37 mg (290 mg, 0.491 mmol) is obtained in a pink solid form with an efficiency of 93%.
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The reactivity of compound 37 was tested on 3'-Urdine monophosphate and followed by capillary electrophoresis. The reagent is stable at -20 °C for at least 1 month.
The following is a list of the active substances in the active substance: The following is a summary diagram:
• Protection of methyl-3-formylbenzoate 38:
Dowex 50WX8-100 (2 g) resin is dissolved in 25 mL of MeOH and 25 mL of trimethyl orthoformiate and agitated for 15 min. After settling, the resin is washed twice with 20 mL of MeOH. This resin is then added to 100 mL of MeOH, 50 mL of CHOM((e) 3 and 7.12 g (43.4 mmol) of methyl-3-formylbenzoate. The solution is agitated for 15 min. The product is filtered on pleated paper before evaporation.
The following are the values of the measurement of the radiation emitted by the vehicle:
• Composed of 40:
The solution obtained is heated to 165°C for 2 h. The mixture is then dissolved in 80 mL of DCM and washed 4 times with 20 mL of water. After drying on MgSO4 and evaporation, the product 40 is isolated with an efficiency of 60% (2.27 g 5.69 mmol).
The following are the main components of the test method: the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test, the test, the test, the test, the test, the test, the test, the test, etc.
• Biotinyl compound 41:
The D-biotin (344 mg; 1.40 mmol) is suspended in 4 mL DMF and 250 mg (1.54 mmol) CDI is added. This solution is left to agitate for 30 min at room temperature. Compound 40 (616 mg; 1.54 mmol) is dissolved in 2 mL DMF and then added gradually to the previous solution. The resulting mixture is left to agitate for 50 min at room temperature. After evaporation, a column purification by flash chromatography (column 30 mm diameter) is performed with 750 mL MeOH-DCM 10%, then 250 mL MeOH-DCM 15%. The fractions corresponding to the 41 mg product are evaporated to give an estimated evaporation of 740 mg of oil at 50%.
The following are the main components of the test method: - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical, - the measurement of the concentration of the test chemical in the test chemical in the test chemical, - the measurement of the concentration in the test chemical in the test chemical, - the measurement of the concentration in the test chemical in the test chemical in the test chemical, - the measurement of the concentration in the test chemical in the test chemical in the test chemical, - the measurement of the concentration in the test chemical in the test chemical in the test chemical in the test chemical, - the measurement in the test chemical in the test chemical in the test chemical in the test chemical in the test chemical in the test chemical in the test chemical in the test chemical in the test chemical in the test, - the measurement in the test chemical in the test chemical in the test chemical in the test chemical in the test chemical in the test, - the measurement in the test chemical in the test in the test chemical in the test in the test chemical in the test in the test in the test in the test in the test in the test in the test in the test in
• Aldehyde compound 42:
The acetal 41 is dissolved in 20 mL of chloroform, then 5 mL of HCl 2N is added. The diphasic mixture is vigorously agitated for 15 min. The organic phase is recovered and dried on anhydrous NaHCO3.
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• Compound hydrazone 43:
The yellow oil obtained after evaporation is crushed with ether until it becomes a beige powder corresponding to product 43 (404 mg; 0.68 mmol) with an efficiency of 66%. A column purification by flash chromatography (column 15 mm diameter) is then carried out on a 150 mg (0.253 mmol) sample with 200 mL of MeOH-DCM. The collected fractions are then evaporated to 144 mg 20% to give a product with an efficiency of 76%.
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• Compound diazo 44:
Hydrazone 43 (100 mg; 0.187 mmol) is dissolved in 4 mL of DMF. MnO2 (200 mg; 2.3 mmol) is then added. After 13 min of stirring at normal temperature, the mixture is filtered onto a millipore containing cellulite (thickness: 2 cm) and molecular sieve into a powder of 3 Å (0.5 cm). The reaction mixture is evaporated dry. The resulting residual oil is washed with ether three times in a row. Compound 44 (290 mg; 0.491 mmol) is obtained as an orange solid with an efficiency of 83%. The following shall be reported for the test chemical:The following are the main components of the test method: - the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, the test method, test method, test method, test method, test method, test method, test method, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test, test,B9); 1, 25 (m, 2H, HB8); and
The product stability is more than 1 month at -20°C.
Example 23 : Synthesis of the para-Cy5-EG3-PDAM:
As already mentioned in example 2, biotin can be replaced by another marker such as Cy5, which shows that the diazo function carried by PDAM can also be linked to this Cy5 marker by means of a polyethylene glycol binding arm.
Synthesis diagram: The I-contray is not represented in formulae 46, 47, 50' and 51. - What?
• Iodide of 2-[4-[N-acetyl-N-phenylamino]buta-1,3-dienyl]-1,2,3,3-tetramethyl[3H]indolium 47:
The mixture of malonaldehyde-bis (phenyl) monochloride 45 (13 g; 50.2 mmol), NaOAc (6.0 g; 69.7 mmol) and 1,2,3,3-tetramethyl[3H]indolium 46 iodide (3.01 g; 10 mmol) in acetic anhydride (50 mL) is heated to 100 °C (50 °F) for precisely 20 min. After cooling, ether (350 mL) is added and the precipitated brown solid is filtered, washed with ether (3 × 100 mL).
The following are the values of the 1H (CDCl3) NEM: δ = 8,64 (d ; 1H ; J= 12 Hz ; 1-H) ; 8,14 (t ; 1H ; J = 16 ; 12 Hz ; 3-H) ; 7,63-7,19 (m ; 9H) ; 6,90 (d ; 1H ; J = 15 Hz ; 4-H) ; 5,82 (t ; 1H ; J = 12 ; 13 Hz ; 2-H) ; 4,06 (s ; 3H ; NCH3) ; (2 ,16 (s ; 3H ; -COCH3) ; 1,74 (s ; 6H ; CH3).
• Bromide of 1- ((5-carboxypentyl)-2,3,3-trimethyl[3H]indolium 50:
The 2,3,3-trimethylindole 48 (10.0 g; 62.8 mmole) and 6-bromohexanoic acid 49 (12.3 g; 62.8 mmole) are mixed without solvent and heated at 110 °C for 12 h under argon. The purple-red paste reaction mixture is washed with ethyl acetate (2 × 60 mL, the paste is crushed with the spatula and the supernatant is settled), then with acetone (50 mL, the paste solidifies). The pink solid is filtered and then vacuum dried (16.0 g; 73 %).
•Compound Cy5COOH 51:
The mixture of iodide 47 (2.5 g; 5.3 mmole), bromide 50 (1.87 g; 5.3 mmole) and NaOAc (1.08 g; 12.1 mmole) in acetic anhydride (11 mL) is heated to 120 °C for 25 min. After cooling, ether (200 mL) is added and the precipitate is filtered and washed with ether (3 × 50 mL). The solid corresponding to product 50' is then dissolved in 100 mL of CH2Cl2 and evaporated. It is dissolved in 15 mL of acetic acid and stirred for 30 min at 120 °C. The precipitate corresponding to product 51 is thus obtained after 200 mL of ether and filtration, with an efficiency of 84% (244 g, 4.71 mmol).
The following are the values of the values of the values of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measur - What?
• Coupling of compound 26 with Cy5COOH 51 (product 52):
To a solution of Cy5COOH 51 (1.5 g; 2.46 mmole) in 15 mL CH2Cl2, add N-methylmorpholine (NMM, 405 μL; 3.68 mmole). The solution is cooled with an ice bath and placed under argon, then isobutyl chloroformate (494 μL; 3.81 mmole) is added. After 10 min of stirring, amine 26 (1.86 mg 4.67 mmole) diluted in 8 mL CH2Cl2 is added. The mixture is left to stir at room temperature for 1 h. Add 20 mL CH2Cl2 and wash with 25 mL NaHCO3 (1 NCO3) three times in a row. After drying on Na2CO3,The solution is filtered to recover the dichloromethane phase which is evaporated. A column-by-column chromatography-flash purification (column 45 mm diameter, fractions of 20 mL)) is performed with 10% MeOH-DCM as the electrolyte. The fractions corresponding to product 52 are collected and then dry evaporated to give a blue solid which is dissolved in CH2Cl2. The product 52 (iodide) is then dissolved in 54 mL of methanol and passed on to an IRA900 (Cl-; 15 g) column of amberlite.The methanol solution collected is evaporated dry to give a sticky oil which is redissolved in CH2Cl2.
• Aldehyde 53:
The 52' acetal is dissolved in 10 mL of DCM and then 10 mL of HCL 2N is added. The solution is left to agitate for 3 h 30 min. After adding 20 mL of DCM, the dichloromethane phase is recovered and then dried on NaHCO3. The product obtained after evaporation is washed with ether to give aldehyde 53 with an efficiency of 90% (1.18 g 1.46 mmol).
• Hydrazone 54:
The solution is agitated at room temperature for 30 min. 8 mL of ether is added; the ether is washed by settling three times in a row and then vacuum dried. 172 mg of ether is obtained (54 mmol; 85 per cent yield) and stored in the freezer.
- Diazo 55:
To a 20 mg (0.243 mmol) solution of hydrazone 54 in 2 mL DMF, 100 mg of MnO2 is added and vigorously shaken for 5 min under argon at room temperature. The suspension is filtered through a layer of cellite (thickness: 2 cm) and molecular sieve into a powder of 3 Å (0.5 cm) and washed with DMF. The solution is evaporated and then crushed with ether. The resulting solid is dried. 18 mg (0.185 mmol; 76%) diazo 55 is obtained. The stability of the reagent is more than 1 month at -20 °C.
The following is the list of active substances:
As already mentioned in example 23, biotin can be replaced by another marker, which shows that the diazo function carried by PMDAM can also be linked to this fluorescein marker by means of a polyethylene glycol binding arm.
The following is a summary diagram:
Compound 72:
The fluorescein-isothiocyanate (250 mg, 0.64 mmol) is solubilised in 1.6 ml of anhydrous DMF with 2% pyridine under argon. Product 69 (0.356 g, 0.81 mmol) dissolved in 1.6 ml of anhydrous DMF is added. It is allowed to react for 3.5 h at room temperature, then DMF is evaporated and taken up again in 25 ml of H2O. Three extractions are made with 50 ml of CH2Cl2, and the aqueous phase is evaporated. 255 mg (48%) of product 72 is obtained.
The following is added to the list of active substances:
The compound 72 (255 mg, 0.31 mmol) is dissolved in 1.5 ml of ethanol at reflux. Lap-toluene-sulfonyl-hydrazine (69.2 mg, 0.37 mmol) is added to 1.5 ml of ethanol and allowed to react for 6 h. The solid is evaporated dry and washed with CH2Cl2, H2O and ether. 18.5 mg (74%) of product 75 is obtained as an orange powder. The frequency range of the measurement is defined as the frequency range of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measurement of the measur
The following substances are to be classified in the same heading as the active substance:
Hydrazone 75 (176 mg, 0.18 mmol) is dissolved in 720 μl of a 10% KOH solution in anhydrous methanol. The solution is left to reflux for 3 h. The solution is allowed to cool and a precipitate appears. The solution is filtered and evaporated dry. The residue is washed with ether and dried. The MRI analysis shows the disappearance of signals at 2.36 and 2.13 ppm (corresponding to the methyls of tosyl and hydrazone) and the appearance of a peak at 1.96 ppm (corresponding to the methyl of diazo).
Example 25: Diazomethyl intermediate allowing for further marking:
It may be interesting to have not a direct marking by the diazomethyl marking reagent also bearing the R2 marking but to proceed in two steps with an indirect marking. In this case the marking reagent including the diazomethyl function is said to be prefunctionalised i.e. it also includes a chemical function capable of subsequent reaction with direct or indirect markers. Prefunctionalisation may be by introducing a reactive covalence function within the marking reagent which may react with an anti-reactive covalence function of the direct or indirect marker. These functions may be constituted by an organic electrophilic chemical function and an organic nucleophilic chemical function, or vice versa.
An example of such a marking strategy is illustrated by the diagram below. - What? In which the labelling reagent includes, in addition to a diazomethyl function, an electrophilic or nucleophilic W1 function capable of reacting with a marker R2 including a complementary W2 function of W1. For example, if W1 is a methyl ketone or aldehyde function, W2 is an alcohol-amine function. In a labelling process of a biological molecule such as a nucleic acid, the nucleic acid is brought into contact with the labelling reagent covering the diazomethyl function and in a further step, the marker W2-R2 reacts with the nucleic acid via the W1 function. One of the uses is for example a process for amplifying a nucleic acid sequence or a signal amplification process.
Example 25.1: Summary of the MeCO-PMDAM: The following is a summary diagram:
The product 85 synthesized in this example allows the marking of natural nucleic acids by the reactivity of the diazomethyl function with phosphate groups and thus introduces a methyl ketone function, which can then be used to introduce a detectable molecule (fluorescent, biotin) with an alcohol group.
The first step is the protection of the terminal amine by Fluorenylmethylformate (Fmoc, 99). The choice of this protective grouping is based on its stability and cleavage conditions. - What?
After the formation of the protected hydrazone 82 by the method used previously (e.g. meta-Fluo-EG3-PMDAM,), the terminal amine is unprotected under mild basic conditions that ensure the stability of the hydrazone.
The test chemical is used to determine the concentration of H2NO-PMDAM in the test medium. The following is a summary diagram:
The product 88, the synthesis of which is described in this example, allows the marking of natural nucleic acids, by the reactivity of the diazomethyl function with phosphate groups, and thus introduces an alkoxyamine function, which can be used subsequently to introduce a detectable molecule (fluorescent, biotin) with a methyl ketone group.
This synthesis is based on the model of the previous one, i.e. the use of precursor 69, Fmoc for the protection of the amine and tosyl for the protection of the hydrazone. The introduction of the alcoholxyamine function (compound 86) is done by the use of the carboxymethoxylamine (commercial) protected by the Fmoc function (Thesis E. Trevisiol, LEDSS Grenoble, 1999).
Example 26: Preparation of PDAM derivatives allowing for signal amplification: Example 26.1: Synthesis of bis-biotinylated markers such as [Bio-EG3]2-PDAM: The following is a summary diagram:
•Reduction of benzene-1,3,5-tricarboxylate of trimethyl 56 to alcohol 57:
The red solution is heated to 40-45°C under argon agitation for 1 hour. After cooling (ice), the excess hydride is carefully destroyed (release of H2) by the addition of water (200 mL) and then HCl 2N (30 mL). The colour changes to light yellow. This solution is extracted with CH2Cl2 (100 mL and then 3 mL), the organic phase is washed with NaHCO3 anhydrous, dried on MgSO4 soil until it evaporates once (11,1 g).
The frequency of the signal is measured at a frequency of 1 Hz (200 MHz, CDCl3): δ = 8,53 (t, 1H, J= 2 Hz); 8,18 (d, 2H, J= 2 Hz); 4,76 (s, 2H); 3,91 (s, 6H); 2,30 (s, 1H).
• Oxidation of alcohol 57 to aldehyde 58:
The solution is left overnight agitated under argon, filtered through a Büchner funnel with a layer of Celite 545, washed with CH2Cl2 and evaporated. The crude solid (4.4 g) is purified by flash chromatography on a silica column (diameter = 50 mm, emulsifier: ethyl acetate/cyclohexane 3/7).
The frequency of the signal is measured at a frequency of 1 Hz.
• Formation of acetal 59:
Aldehyde 58 (3.21 g; 14.4 mmol) is dissolved in 30 mL of methanol and 6.0 mL of TMSCI is added.
The solution is diluted with 200 mL of CH2Cl2, and stirred with 1 M NaHCO3 (100 mL) (careful CO2 release). The two phases are separated, the aqueous phase is extracted three times with CH2Cl2 (25 mL), the organic phases are combined, the solvent is dried on MgSO4 and then evaporated. The frequency of the signal is measured at a frequency of 1 Hz (MHz, CDCl3): δ = 8,63 (t, 1H, J = 2 Hz); 8,29 (d, 2H, J = 2 Hz); 5,45 (s, 2H); 3,93 (s, 6H); 3,32 (s, 6H).
• Hydrolysis of diester 59 to diacid 60:
The dissolved dister 59 (3.18 g; 11.9 mmol) is dissolved in 10 mL of THF and then a solution of KOH (2.0 g, 85% capsule) is added to 10 mL of methanol. After 15 min at room temperature, the solvents are evaporated. The residue is dissolved in H2O (50 mL). H3PO4 (approximately 2.5 mL, 85%) is added to pH 3 and the white precipitate is filtered on the frittate (#3), washed with water and vacuum dried.
The frequency range of the measurement is defined as the frequency range of the measurement.
• Trifluoracetamide 62:
Diamine 61 (66 g; 0.30 mol) is dissolved in 250 mL of CH2Cl2 and then ethyl trifluoroacetate (11.8 mL, 0.10 mol) is added drop by drop for 5 min at 10 °C under argon agitation. After 15 min at room temperature, the solution is transferred to a decanting ampoule, washed with H2O (3 × 100 mL), dried on MgSO4 and evaporated. 22.4 g (71%) of monoamide 62 of about 85 % purity (determined by F19 RMN) is obtained. This compound is stored at -20 °C and used without purification.
The frequency of the signal is measured at a frequency of 1 Hz (MHz, CDCl3): δ = 3,5-3,6 (m, 12H); 3,42 (t, 2H, J= 6 Hz); 2,75 (t, 2H, J= 6 Hz); 1,81 (quantiplet, 2H, J= 6 Hz); 1,67 (quantiplet, 2H, J= 6 Hz); 1,30 (s. wide, 2H). The frequency of the measurement shall be determined by the measurement of the frequency of the measurement.
•Compound 63:
The mixture is heated to 55-60 °C under argon agitation for 30 min. Initially a complete dissolution of the material is observed and then a mass uptake with precipitation of a white solid (CO2 evolution). The amine (oil) is added using 5 mL of CH2Cl2 to rinse and the mixture is heated to 55-60 °C for 3 h. The DMF (< 1 mmHg) is evaporated under vacuum and the residue is agitated with CH2Cl2 (700 mL) and 2N HCl (100 mL).After filtration of the two phases through a layer of Cellite 545, the phases are separated, the aqueous phase is extracted with CH2Cl2 ((15 × 100 mL), the organic phases are collected, dried on anhydrous NaHCO3 and MgSO4, then the solvent is evaporated. The oily residue is crushed with 150 mL of ether to obtain a suspension. The paste-like solid is difficult to filter. The supernatant is decanted and the ether washing is repeated. The following is the value of the value of the measurement:The first two are: 2H, J=6 Hz); 2.75 (t, 2H, J=6 Hz); 1.81 (quantiplet, 2H, J=6 Hz); 1.67 (quantiplet, 2H, J=6 Hz); 1.30 (s. wide, 2H).
• Composed of 64:
After cooling, the solvent is evaporated. The residue is dissolved in methanol (20 mL) and passed onto a Dowex 21K anion exchange resin column [height 12 cm × diameter 35 mm, OH-form obtained by pre-washing with NaOH 1N (1.5 L) then H2O (1.5 L) then methanol (1.5 L) ]. The free trifluoride ion 64 is passed into the first fractions with 200 mL of methanol. After the treatment, the residue is triturated with 50 mL of ether.
The frequency range of the signal is defined as the frequency range of the signal, which is the frequency range of the signal, and the frequency range of the signal.
•[Bio-EG3]2-acetate 65:
To a suspension of diacid 60 (120 mg; 0.500 mmol) in dichloroethane (5 mL), add carbonyldiimidazole (225 mg, 90%, 1.25 mmol) and heat to 55-60 °C for 30 min under argon agitation. Add amine 64 (550 mg; 1.23 mmol) and heat the solution to 55-60 °C for 6 h. After evaporation, pass on a silica column (diameter: 25 mm, electrolyte: methanol 15-30% in CH2Cl2).
The mean of the measurements is given by the mean of the measurements of the two samples, which are given in Table 1 of this Annex.
• [Bio-EG3]2-aldehyde 66:
Acetal 65 (413 mg; 0.376 mmol) in solution in methanol is treated with HCl 2N (0.5 mL). The frequency range of the signal is defined as the frequency range of the signal, which is the frequency range of the signal, and the frequency range of the signal.
The following shall be added to the list of substances:
Aldehyde 66 is converted to diazomethane 67 by the method used for the preparation of diazomethane (Example 1). The stability of the reagent is more than 1 month at -20 °C.
The following is the list of active substances: The following is a summary diagram:
Compound EG3-acetophenone 76:
The 3-, 6-, 9-trioxa-1, 11-undecanedioic acid (EG3, 12.64 ml, 74 mmol) is dissolved in 80 ml of DMF anhydrous under argon and cooled in an ice bath. The dicyclohexylcarbodiimide (DCC, 11.45 g, 55.5 mmol) is then dissolved in 20 ml of DMF anhydrous and added slowly. After 30 min, the 3-aminoacetophenol (5.0 g, 37 mmol) is added and then allowed to react for 1 h at room temperature under argon. The DMF is evaporated under vacuum and 70 ml of CH2CL2 are added. The solution is filtered and extracted with 3x25 ml of 1 ml of organic acetic acid. The combined and mixed phases with the 25 ml of CH2 are mixed in water, thus producing a dry solution of sodium anhydride (OH 7.74 g).
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Compound (NH2) 7-EG3-acetophenol 77:
After 30 min, add this solution to a solution of the commercial dendrimer Starburst PAMAM Dendrimer, Generation 1 (Aldrich, St Quentin Fallavier) (1 g, 0.71 mmol, in 5 ml of methanol), slowly and under strong agitation. React for 1 h at room temperature and evaporate. The residue is taken up again in 10 ml of CH2CL2 and extracted twice with 30 ml of 1% acetic acid.
The active substance is Biot7-EG3-acetophenone 78:
D-Biotin (1.73 g, 7.08 mmol) is soluble in 80 ml of anhydrous DMF under argon, and the solution is cooled on ice. N-methylmorpholine (NMM, 856 μl, 7.7 mmol) and isobutyl chloroformate (1022 μl, 7.7 mmol) are added successively. After 30 min, product 77 (1.13 g, 0.7 mmol, in 5 ml of methanol) is added and allowed to react for 3 h on ice and vacuum.
The following shall be indicated in the table:
The compound 78 (300 mg, 0.09 mmol) is dissolved in 10 ml of absolute ethanol at reflux. Hydrazine monohydrate (20 ml, 0.40 mmol) is added and allowed to react for 3 h at reflux. After cooling, a precipitate is formed, which is filtered, washed with ether and vacuum dried.
The following shall be added to the list of active substances:
Hydrazone 79 (100 mg, 0.03 mmol) is dissolved in 5 ml of anhydrous DMF at 70°C. It is allowed to return to room temperature and MnO2 (31 mg, 0.36 mmol) is added. It is allowed to react for 10 min and the manganese oxide is removed by filtration on a frittata with cellulite (0.5 cm and molecular sieve powder (0.5 cm). The filtrate is evaporated dry, washed in ether and vacuum dried. This results in 78 mg (78%) of product 80.
Err1:Expecting ',' delimiter: line 1 column 679 (char 678)
Example 27: Marking and two-step fragmentation of DNA amplifiers with meta-BioPMDAM:
DNA amplicons were prepared by PCR amplification according to the protocol described in example 5. Two marking reactions were performed.
a. Marking and fragmentation in two stages:
Add 10 μL of meta-BioPMDAM (100 mM in DMSO) and 77 μL of Dnase/Rnase free water to 10 μL of PCR. Incubate the solution for 10 min at 95°C. Then add 3 μL of HCl at 0,1M and incubate the solution for 10 min at 95°C.
b. Marking and fragmentation in one step:
10 μL of PCR is followed by 10 μL of meta-BioPMDAM (100 mM in DMSO), 5 μL of HCl at 0.1 M and 75 μL of Dnase/Rnase free water. The solution is incubated for 30 min at 60°C. The rest of the protocol is the same as in example 9.
Results:
Etude comparative du marquage et de la fragmentation en deux étapes distinctes et en une seule étape
a. Marquage et fragmentation en deux étapes 99,5 14129 624 22,7
b. Marquage et fragmentation en une étape 98,9 4431 667 6,6
As shown in Table 13, the results obtained with the one-step protocol are satisfactory, those obtained with a marking and a two-step fragmentation are even better. This example shows that the marking and cleavage steps can be dissociated to improve marking according to the target used.
Example 28: Marking and fragmentation of DNA amplicons in different reaction formats:
DNA amplifiers were prepared by PCR amplification according to the protocol described in example 5.
a. Marking and fragmentation in a 250 μL format:
Add 75 μL of meta-BioPMDAM (100 mM in DMSO) and 102.5 μL of Dnase/Rnase free water to 50 μL of PCR. Incubate the solution for 25 min at 95°C. Then add 22.5 μL of HCl at 0,1 M and incubate the solution for 5 min at 95°C.
b. Marking and fragmentation in a 200 μL format:
Add 75 μL of meta-BioPMDAM (100 mM in DMSO) and 52.5 μL of Dnase/Rnase free water to 50 μL of PCR. Incubate the solution for 25 min at 95°C. Then add 22.5 μL of HCl at 0.1 M and incubate the solution for 5 min at 95°C.
c. Marking and fragmentation in a 150 μL format:
Add 75 μL of meta-BioPMDAM (100 mM in DMSO) and 2.5 μL of Dnase/Rnase free water to 50 μL of PCR and incubate the solution for 25 min at 95°C. Then 22,5 μL of 0,1M HCl is added and the solution is incubated for 5 min at 95°C.
The remainder of the protocol is identical to that in Example 9.
Results:
Marquage et fragmentation selon différents formats
100,0 5606 549 10,2
99,4 5886 557 10,6
99,4 6800 537 12,7
The results obtained in terms of signal and percentage of homology are very satisfactory in all cases of figures. This example shows the flexibility of the reactive format of the marking protocol to accept different volumes and in particular different volumes of amplification products.
Example 29: Comparison between a protocol using a purification step before fragmentation and a protocol using a purification step after fragmentation:
DNA amplifiers were prepared by PCR amplification according to the protocol described in example 5.
a. Marking, purification and subsequent fragmentation of DNA amplifiers:
10 μL of PCR is added to 10 μL of meta-BioPMDAM (100 mM in DMSO) and 80 μL of Dnase/Rnase free water. The solution is incubated 10 min at 95°C. Then purification is performed according to the protocol described in example 9. The composition of the hybridization buffer and the rest of the protocol are identical to that of example 9.
b. Fragmentation marking and then purification of DNA amplicons:
Add 10 μL of meta-BioPMDAM (100 mM in DMSO) and 77 μL of Dnase/Rnase free water to 10 μL of PCR and incubate the solution for 10 min at 95°C. Then add 3 μL of HCl at 0,1 M and incubate the solution again for 10 min at 95 °C. The rest of the procedure is the same as in example 9.
Results:
Comparaison entre protocole utilisant une étape de purification avant fragmentation et protocole utilisant une étape de purification après fragmentation
a. Purification avant fragmentation 98,9 6256 473 13
b. Fragmentation avant purification 96,1 6066 556 11
This result, shown in Table 15, shows that the purification step can be introduced between the marking and fragmentation stages, and that the introduction of purification between the marking and fragmentation stages enables denaturation during cleavage to be achieved and all the marked amplicon fragments to be hybridized on the chip.
The following is the list of substances which are to be used in the preparation of the test chemical: The following is a summary diagram:
• Protection of aldehyde:
5 g (25.5 mmol) of 2,4-dinitrobenzaldehyde is dissolved in 250 mL of toluene, and 20 mL of ethylene glycol and 150 mg of para-toluenesulfonic acid are added. The oil is heated by reflux by recovering water in a Dean-Stark system for 6 h. 150 mL of EtOAc and 100 mL of H2O are treated.
The following is a list of the most commonly used methods for the determination of the concentration of a substance in a sample:
•Reduction of the dinitryo 100 derivative:
The protected 2-4-dinitrobenzaldehyde (6.4 g; 25.5 mmol) is dissolved in an ethanol-water mixture (6/1), then 2 equivalent Na2S nonahydrate (12.3 g; 51.1 mmol) is added. The reaction mixture is then heated for 30 min. Evaporation and then dichloromethane extraction are performed. After drying and filtration, the reaction medium is evaporated to an oil, which is directly purified on silica column (cyclohexane-ethyl acetate 60/40). Compound 101 is isolated with an efficiency of 45%.
It is composed of 101: F 58-60°C. - RMN 1H (200 MHz, CDCl3): 7,49 (d, 1Haro, J= 2 Hz, H3); 7,09 (d, 1Haro, J= 2 Hz, H6); 6,80 (dd, 1Haro, J= 2 Hz, J= 6 Hz, H5); 6,27 (s, 1H, CH) ; 3,99-3,97 (m, 4H, CH2-CH2).
• Coupled with biotin:
D-biotin (1,0 g; 4,1 mmol) is soluble in 20 mL of anhydrous DMF and 600 μL of N-methylmorpholine. Isobutyl chloroformate (700 μL; 5,5 mmol) is added under argon by cooling in an ice bath. It is stirred for 5 min, then 1 g (4.75 mmol) of the compound 101 and 500 μL of N-methylmorpholine is added. The solution is kept stirred at room temperature for 4 h, then evaporated dry. The resulting oil is passed directly onto a silica column with MeOH-DCM 7 % and then 10 % as the elution solvent. The result is 102 (1,1 g 2,);52 mmol) is obtained with a yield of 62%. The following are the values of the values of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of the measurements of theThe test chemical is a chemical compound with a specific gravity of 10 μm.
• Deprotection of acetal:
The product 102 (768 mg; 1.76 mmol) is placed in suspension in 25 mL of THF. Everything dissolves after adding 4 mL of H2SO4 2N. It is stirred for 2 h. Evaporates and then rinsed and washed with water on a frittate.
The following equation is used for the calculation of the mean value of the measurement of the radiation emitted by the vehicle:
•Formation of hydrazone 104:
When hydrazine is added, everything dissolves and the solution immediately turns orange. A precipitate is formed after 5 minutes. It is heated under agitation for 1 hour. The precipitate is filtered on frit and then dried. The product 104 (700 mg; 690 mmol) is obtained with an efficiency of 98%.
The following equation is used for the calculation of the mean value of the measurement of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of the measured values of
• Formation of diazo 105:
The compound 104 (200 mg; 0.492 mmol) is dissolved in 8 mL of DMF. 400 mg of MnO2 is added. It is vigorously shaken for 10 min. Filtered on a millipore containing cellulite (thickness: 2 cm) and molecular sieve into a powder of 3 Å (0.5 cm). Dry evaporated and then washed with ether. Filtered again on a millipore. Compound 105 (180 mg; 0.445 mmol) is obtained as an orange powder with an efficiency of 98%.
The following equation is used for the determination of the concentration of the active substance in the feed additive:
The reactivity of compound 105 was tested on 3'-Urdine monophosphate followed by capillary electrophoresis. The conditions of analysis are as in example 6.1. The results show a half-reaction time of 45 minutes.
Example 31: Marking and fragmentation of DNA amplifiers with the marking reagent 2-nitro-para-BioPDAM:
The 2-nitro-para-BioPDAM derivative was obtained according to the reaction pattern described in Example 30. The DNA amplifiers were prepared by PCR amplification according to the protocol described in Example 5.
a. Marking by the reagent 2-nitro-para-BioPDAM:
Add 2 μL of 2-nitro-para-BioPDAM (100 mM in DMSO), 5 μL of HCl at 0,1 M and 83 μL of Dnase/Rnase free water to 10 μL of PCR. Incubate this solution for 30 min at 60°C.
b. Marking by the reagent meta-bioPMDAM:
Add 2 μL of meta-BioPMDAM (100 mM in DMSO), 5 μL of HCl at 0.1 M and 83 μL of Dnase/Rnase free water to 10 μL of PCR. Incubate this solution for 30 min at 60°C.
The remainder of the protocol is identical to that in Example 9.
The result:
: Etude comparative du marquage d'ADN par le dérivé 2-nitro-BioPDAM vis-à-vis du dérivé -BioPMDAM
100,0 24392 899 27,1
98,9 21883 774 28,3
The 2-nitro-para-BioPDAM reagent used for DNA marking gives interesting results in terms of marking intensity and percentage of homology.
Example 32: Insertion of a double bond between the diazomethyl function and the phenyle nucleus, removal of the DAM and synthesis of a molecule particularly suited to this removal:
The aim is to remove the diazomethyl function (DAM) from the aromatic structure to minimise the effect of steric congestion during phosphate alkylation and also during hybridisation of the tagged nucleic acid with its complementary sequence.
The following is a summary diagram:
For the aldol reaction to form (para-methoxycarbonyl) -styrylmethylketone 89, ethyl acetate is used for the high acidity of the methylene protons, which facilitates attack of the formyl group, with subsequent elimination of H2O (favoured by conjugation of the double bond with the aromatic ring) and decarboxylation by hydrolysis due to the base medium. The final product para-Bio-EG3-SMDAM 90 has two additional carbons between the diazomethyl and the aromatic ring, which limits possible sterile problems, while maintaining the stabilization of the diazomethyl by the aromatic system by conjugation.
Example 33: Capture and detection of a nucleic acid on a solid medium with diazomethyl groups:
The reactivity of a resin with diazomethyl groups was studied to determine its ability to bind nucleic acids.
4- ((Diazomethyl) phenoxymethyl-polystyrene (reference 17338, Fluka) is a resin described for its ability to bind carboxylic groups, particularly those found in proteins (G. Bhalay, A.R. Dunstan, Tetrahedron Lett. 39, 7803 - 1998), but it is not described for its ability to bind DNA molecules. We tested the possibility of capturing nucleic acids with this reagent, and revealing them by a colorimetric test.
The experiment is performed with some of the reagents present in the HLA-DR oligo-detection kit (reference 33 202, bioMérieux, France basic principle described in patent EP 549 776-B1), allowing the detection of PCR amplified nucleic acids in microplaques, by colorimetric reading. In the experiment described, the PCR-produced nucleic acids are reacted simultaneously with the test resin, as well as with a para-Bio-EG3-PDAM molecule, the synthesis of which is described in example 20. If the DNA reacts with the streptomycin functions on the two compounds, it will reveal the color after washing, and the elimination of the molecules present in the test resin, making it possible to create a colorless reaction with the active substance Lactoferrin (Pyridine) which is a colorless dye, making it possible to create a colorless reaction with the active substance 1 pptx, which is a colorless dye.
Example 33.1: DNA capture and detection:
Incubate 10 mg of resin for 30 minutes at 60°C with 50 μL of PCR as described in example 5 in 400 μL of pure water (Sigma) with 5 μL of para-Bio-EG3-PDAM. Wash this resin with 500 μL of PBS tween buffer (Kit Reagent Color 0 HLA, PBS pH 7.0; Tween 1%, BND 0.2 g/L Ciproflaxacin 0.01 g/L). Then resuspend the resin in 100 μL of PBS Tween and 250 μL of streptavidine hybridization buffer (BPS pH 7.0 TWEEN 0.5%) with streptavidine HRP (SUL-911, MOLECARB, PROGENE,The reaction mixture is incubated for 30 min at room temperature, then washed three (3) times with 500 μL of PBS tween buffer, and incubated at room temperature in the presence of chromogenic reagent (1 Color 1 tablet, orthophylene diamine hydrochloride, diluted in 5 mL of Color 2 buffer, Sodium phosphate 100 mM, citric acid 50 mM, H2O2 0.03%). After 20 min incubation in the dark, the reaction is blocked by 50 μL of H2SO4 (1.8 N) over the reaction medium.
Example 33.2: Nucleic acid-free witness:
Incubate 10 mg of resin for 30 minutes at 60°C in 425 μL of pure water (Sigma) with 5 μL of para-Bio-EG3-PDAM, then wash this resin with 500 μL of PBS buffer, and then treat the sample in the same manner as described in example 33.1.
Example 33.3: Witness with PCR performed without targets:
Incubate 10 mg of resin in 400 μL of pure water with 5 μL of para-Bio-EG3-PDAM for 30 minutes at 60°C, with 50 μL of PCR performed with a volume of 25 μL of pure water in place of the volume of genomic DNA described, then wash this resin with 500 μL of PBS buffer, and then treat the sample in the same manner as described in example 33.1.
Example 33.4: Witness with PCR without revealing molecule:
Incubate 10 mg of resin with 50 μL of PCR in 400 μL of pure water for 30 minutes at 60°C. Wash this resin with 500 μL of PBS buffer and treat the sample in the same manner as described in example 33.1.
Example 33.5: Witness with uncaptured nucleic acid:
Incubate 10 mg of resin for 30 minutes at 60°C with 400 μL of pure water and 5 μL of para-Bio-EG3-PDAM. Wash this resin with 500 μL of PBS tween buffer (kit's Color 0 HLA Reagent, PBS pH 7.0; Tween 1%, BND 0.2 g/L; Ciproflaxacin 0.01 g/L). Resuspend the resin in 100 μL of PBS Tween and 250 μL of streptavidine hybridization buffer with streptavidine HRP diluted at 1/10000th. Add 50 μL of DNA prepared as follows to this preparation:
Add 5 μL of para-Bio-EG3-PDAM and 70 μL of pure water to 25 μL of DNA from a PCR prepared as described in Example 5. Incubate this mixture for 30 min at 60°C and then remove excess marker by subjecting the preparation to a QIAquick (Nucleotide Removal Kit, Qiagen, Hilden, Germany) column purification following the protocol recommended by the supplier, with final elution in a volume of 50 μL.
The reaction mixture is incubated at room temperature for 30 min and then treated according to the process described in Example 33.1.
Results
Etude de la réactivité d'une résine portant des groupements diazométhyle
527
249
261
264
249
In Table 17, a high colourimetric value indicates a high concentration of enzymes in the reaction medium, corresponding to a high presence of nucleic acid carrying biotin derivatives. The controls indicate that the signal is not due to non-specific adsorption of DNA on the ball, to a reaction of para-Bio-EG3-PDAM on the resin, or to adsorption of streptavidin HRP on the resin, but to the presence of covalently captured DNA and marked by para-Bio-EG3-PDAM.
Example 34: Marking of a PCR product to allow its capture and detection in a micro-plate:
This example shows the possibility of labelling a DNA molecule with a single type of molecule carrying a diazomethyl function, so as to capture and detect this nucleic acid in a single step on a microplate.
The experiment is performed with some of the reagents present in the HLA-DR oligo-detection kit (reference 33 202, bioMérieux), which allows the detection of PCR amplified nucleic acids in microplates, by colorimetric reading. In the experiment described, the para-Bio-EG3-PDAM, whose synthesis is described in example 20, is reacted with nucleic acids produced by PCR. The DNA reacts with the diazomethyl groups of the molecule, and is equipped with biotin grafted onto its surface.
Example 34.1: Capture and detection of DNA from PCR on micro-plate:
After marking, the DNA is purified on a QIAquick column (Nucleotide Removal Kit, Qiagen, Hilden, Germany) according to the protocol recommended by the supplier, and the final effluent is collected in 50 μL buffer hour (Tris EDTA 10 mM, 8.5 pH). V (20) μL of this effluent is diluted in 180 μL buffer PEG (0.1 M Na-EG3-PDAM) or 0.5 M NaCl (0.65% Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween Tween T
Example 34.2: Making witnesses:
Simultaneous controls shall be carried out as follows:
- A. Marking witness without DNA:
Ninety (90) μL of pure water and 10 μL of para-Bio-EG3-PDAM are incubated for 30 min at 60°C. The reaction mixture is then treated in a similar manner to the process described in Example 34.1.
- B. Marking witness without markers:
10 μL of DNA obtained by PCR amplification, as described in Example 5, are incubated for 30 min at 60°C in 90 μL of pure water.
All the bars are then washed three times with 100 μl of PBS Tween buffer (kit's Color 0 HLA Reagent, PBS pH 7.0; Tween 1%, BND 0.2 g/L; Ciproflaxacin 0.01 g/L) and the presence of streptavidine HRP is revealed by adding 100 μl of the chromogenic reagent (1 Color 1 tablet, Orthophenylenediamine hydrochloride, diluted in 5 mL of Color 2 buffer, Sodium Phosphate 100 mM, Citric Acid 50 mM, H2O2 0.03%), incubated in the dark for 20 min, the reaction being then blocked by 50 μL of H2SO4 (1.8 N Reactive Color3).
Results:
Détection d'ADN capturé et marqué par du -Bio-EG3-PDAM
A - ADN marqué 1382 152
B - ADN non marqué 178 136
C - Sans ADN 140 192
The experiment, as shown in Table 18, therefore shows that DNA marked by para-Bio-EG3-PDAM can be captured and detected in a single step in a microplate well. As indicated by the reaction controls, the signal generated is due solely to DNA and is not the result of non-specific adsorption of the nucleic acid on the microplate wall, or on streptavidine, or non-specific reaction of streptavidine HRP on the unmarked DNA, or on the microplate plastic.
Example 35: Double marking of a PCR product to allow its capture and detection on a solid micro-plate type support:
This example shows the possibility of labelling with two molecules, carrying diazomethyl functions, and in one step, a DNA molecule, in order to capture and detect it on a microplate.
The experiment is carried out with some of the reagents present in the HLA-DR trace detection kit, which allows the detection of nucleic acids amplified by PCR in microplates by simple colorimetric reading. The following substances are to be classified in the same category as the active substance: If the DNA reacts with the diazomethyl functions present on both compounds, it can bind to a medium carrying antibodies to pyrene, and it can be detected with a streptavidin molecule associated with a horseradish peroxidase.
Example 35.1: DNA double-marking and detection on microplate:
Antipyrene antibodies were adsorbed onto eight (8) Maxisorp wells by incubating overnight at room temperature 100 μL of a 1.1 μL solution of antipyrene antibodies diluted in 100 μL of bicarbonate buffer (0.05M pH 9.6). Such antibodies, called Rabbit antipyrene (YSRT-AHP236) are available from Accurate Chemical & Scientific (Westbury, New York, USA).
10 μL of DNA obtained by PCR amplification, as described in example 5, was then marked by incubation for 30 min at 60 °C in 40 μL of pure water (Sigma), 10 μL of para-Bio-EG3-PDAM, 2 μL of PDAM (P-1405, 1-pyrenyldiazomethane, Molecular Probes, Eugene, OR, USA) and 38 μL of DMSO. After labelling, the DNA was purified using a QIAquick kit (QIAGEN) and the final effluent was collected in 50 μL of EB buffer (Tris EDTA 10 mM, pH 8.5). Twenty (20) μL of this effluent was diluted in 180 μL of PEG buffer (0.1 M Na PO3; 0.5 M NaCl; 0.65 % Tween 20 ; 0.14 mg/ mL Salmon (Gibco) sperm DNA; 2% PEG 4000) plus streptavidine HRP (S-911, MECULAR PROBES, EUGENE, OR, USA) diluted to 1/ 10,000 (100) μL of this preparation were either incubated for 1 hour at 37°C in a Maxis adsorbent barrel, or in an unsorbed adsorbent barrel, then adsorbed.
Example 35.2: Making of witnesses:
The controls were simultaneously made as follows:
- A. Marking test with para-Bio-EG3-PDAM alone:
Ten (10) μL of DNA obtained by PCR amplification, as described in example 5, are marked by incubation for 30 min at 60°C in 90 μL of pure water with addition of 10 μL of para-Bio-EG3-PDAM. After marking, the DNA is purified using a QIAquick kit and the final effluent is collected in 50 μL of EB buffer. Twenty (20) μL of this effluent is diluted in 180 μL of PEG buffer with addition of streptavidine HRP diluted at 1/10 000. One hundred (100) μL of this preparation is then incubated for 1 hour at 37°C, either in an adsorbed Maxis or in an unadsorbed Maxis barrel well.
- B. Marking witness with PDAM alone:
After marking, the DNA is purified using a QIAquick kit and the final effluent is collected in 50 μL of EB buffer. Twenty (20) μL of this effluent is diluted in 180 μL of PEG buffer with streptavidine HRP diluted at 1/10 000. One hundred (100) μL of this preparation is then incubated for 1 hour at 37°C, either in an adsorbed Maxisorptor barrel well or in an unadsorbed puxis.
- Witness without a mark:
Ten (10) μL of DNA obtained by PCR amplification, as described in example 5, are incubated for 30 min at 60°C in 100 μL of pure water. After marking, the DNA is purified using a QIAquick kit and the final eluate is collected in 50 μL of EB buffer. Twenty (20) μL of this eluate is diluted in 180 μL of PEG buffer with streptavidin HRP diluted to 1/10 000. One hundred (100) μL of this preparation is then incubated for 1 hour at 37°C, either in an adsorbed Maxisorp barrel well or in an unadsorbed well.
The rods are then washed with 100 μL of PBS Tween buffer (color 0) three times and the presence of streptavidine HRP is then revealed by adding 100 μL of chromogenic reagent (Color 2), incubated for 20 min in the dark, the reaction being then blocked by 50 μL of H2SO4 (Color 3).
Results:
Double marquage de l'ADN par le PDAM et le -Bio-EG3-PDAM
348 16
44 19
68 12
75 19
The result in Table 19 clearly shows an important signal resulting from the capture of DNA in the wells by antipyrene antibodies, as well as the simultaneous marking of the DNA by HRP streptavidin, which has become fixed. As shown by the absence of a signal at the control level, this detection is specific to the marked DNA, and is not due to non-specific adsorption of the DNA or HRP streptavidin on the plastic, or non-specific binding of the enzyme on the captured DNA. This example therefore shows that it is possible to achieve double marking of DNA in a single step, as this double marking can be used to capture and detect it simultaneously.
Example 36: Marking of a PCR product to allow simultaneous capture and detection by complementary nuclear probes:
This experiment demonstrates that it is possible to detect specific DNA by capturing such DNA on a solid surface using the reactivity of the diazomethyl function to a phosphate group of DNA.
The experiment is performed with some of the reagents present in the HLA-DR oligo-detection kit (reference 33 202, bioMérieux, France), which allows the detection of nucleic acids amplified by PCR in microplaques, by colorimetric reading. In the context of the described experiment, para-Bio-EG3-PDAM is reacted on nucleic acids produced by PCR. The DNA reacts with the diazomethyl functions of the molecule, and is equipped with biotines grafted on its phosphates.
Example 36.1: Specific DNA capture and detection on microplate:
The 10 μL DNA obtained by PCR amplification is double-labelled by incubating it for 30 min at 60°C with 20 μL para-Bio-EG3-PDAM. After labelling, the DNA is purified on a QIAquick column (Nucleotide Removal Kit, Qiagen, Hilden, Germany) according to the supplier's recommended protocol and the final excretion is collected in 50 μL of buffer EB (Tris-HCl 10 mM, pH 8.5). Eighty-five (85) μL of the excretion mixture is denatured by 8.5 μL of reagent R4 (NaOH 2N) for 5 minutes at room temperature, and the solution is then neutralized by 8.5 μL of reagent R5 (N2 acid).Add 850 μL of hybridization buffer (R6 - Tris-HCL 10 mM, pH 7.0 BND 0.2 g/ L, Ciproflaxacin 0.01 g/ L) and 85 μL of detection oligonucleotide (R7 - Sodium Phosphate 4 mM, Potassium Phosphate 1 mM, pH 7.0, Bovine Albumin Serum 0.1% Phenol 0.5%) to the mixture. One hundred (100) μL of this preparation is deposited either on the control of an R1 bar provided with the kit (captured by a consensus sequence of the amplified gene), or positive on a streptomycin 8 co-plate (reference 95029263, Compact System, Helsinki, Finland), or on a control plate (Nuncor Lab, Maxis, Denmark).
At the same time, the same hybridisation reaction is carried out on a dilution in EB buffer to the tenth and one hundredth of the DNA preparation to test the sensitivity of the technique.
Example 36.2: Making of witnesses:
The controls were simultaneously made as follows:
- Compared to the HLA-DR kit:
Ten (10) μL of DNA obtained by PCR amplification, as described in example 5, are incubated for 30 min at 60 °C with 20 μL of para-Bio-EG3-PDAM. After marking, the DNA is purified on a QIAquick column, and the final eluate is collected in a volume of 50 μL of EB buffer. The 45 μL of eluate is denatured by 4.5 μL of reagent R4 at room temperature for 5 min, then the solution is neutralized by 4.5 μL of reagent R5. 450 μL of hybridization buffer R6 and 45 μL of detection oligonucleotide are added to the mixture. Either (100 μL) of this preparation is deposited on a positive control panel with R1 (complete with 8 g) on a Maxis amplified seed test plate (see section 4.8).
- B - Hybridisation on DNA not hybridising at the specific probe:
10 μL of DNA obtained by PCR amplification, as described in Example 5, are incubated for 30 min at 60°C with 20 μL of para-Bio-EG3-PDAM. The sample is then treated in the same manner as described in Example A.
- Witness without DNA:
10 (10) μL of reagent R6 (hybridization buffer) and 100 μL of reagent R7 (detection oligonucleotide) are deposited either on the positive control of an R1 bar provided with the kit, on a streptavidin plate or on a Maxisorp-type control plate.
All bars of the above protocols are incubated for one and a half hours at 37°C, then washed three times with 100 μL of PBS Tween buffer (HLA colour 0 reagent) and then the presence of the specific detection probe is revealed by addition of 100 μL of the chromogenic reagent (Tween Colour 2, PBS pH 7.0 ; Tween 1%, BND 0.2 g/L ; Ciproflaxacin 0.01 g/L), incubated for 20 min in the dark, the reaction being then blocked by 50 μL of H2SO4 (1.8 N Colour 3 reagent). The absorbance of the reaction medium is then measured at 492 nm.
Results:
Détection spécifique sur microplaque d'un ADN issus de PCR
NA NA
NA NA
The results of Table 20 indicate an excellent amplification of the target for use in a diagnostic setting.This example shows that marking at the phosphate group level allows DNA capture and does not prevent specific hybridization on it.
Example 37: Capture and amplification of DNA from bacterial lysate and marked with para-Bio-EG3-PDAM:
This example shows that it is possible to capture and amplify bacterial DNA using a capture based on the reactivity of the diazomethyl function on nucleic acid phosphate.
In this case, the nucleic acids contained in bacterial lysate and marked with para-Bio-EG3-PDAM are captured on magnetic balls coated with streptavidine, which are then magnetized to remove cellular residues from the reaction medium, which may inhibit the subsequent PCR amplification. The amplification products were analysed by passing on DNA chips.
The bacterial DNA is obtained by lysis of cells contained in a culture of Mycobacterium tuberculosis. The lysis is carried out by mechanical lysis. More specifically, it is carried out by sonication, the treated liquid sample containing glass beads. Such a process is already well described by the Applicant in her patent application WO-A-99/15621, with regard to beads, and in her patent application WO-A-00/60049, with regard to sonication. Sonication can also be carried out using a liquid bath. However, other techniques, known to the trade, may be used such as those described in US-A-5,902,746 and patent applications WO-A-98/54306 and WO-A-00/05338.
The bacterial DNA was quantified by Picogreen (P-7589; Molecular Probes, Eugene, OR, USA) using the protocol described by the supplier at a concentration of 107 copies per μL.
10 μL of lysate are incubated in the presence of 20 μL of para-Bio-EG3-PDAM for 30 minutes at 60°C. At the same time, 10 μL of lysate are incubated in 20 μL of pure water (Sigma) under the same conditions.
The reaction medium is then purified on a QIAquick column (Nucleotide Removal Kit, Qiagen, Hilden, Germany).
The marked DNA fragments are then captured on Dynal (Dynabeads M-280 streptavidin; reference 112.05; Dynal Biotech ASA, Oslo, Norway) magnetic balls, which are prepared according to the following protocol: Ninety (90) μL of Dynal balls are washed twice with 200 μL of pure Free (Sigma) water, then taken up by 200 μL of PEG buffer (0.1 M Na PO4, pH 7.5 M NaCl; 0.65 % Tween 20 ; 0.14 mL of Hareng sperm DNA (Reference 15634-017, GibcoBRL) ; 2 % PEG 4000) and incubated 30 min at 37°C. They are then washed twice with 200 μL of PBS 1X Tween 20 0.5% buffer, then finally taken up by 90 μL of the same buffer.
Incubate 10 μL of the marked or unmarked DNA elulate for 5 min at room temperature with 40 μL of PEG buffer and 2,5 μL of the magnetic bead preparation described above.
The balls are then washed three times with 200 μL of PBS 1 X tween 0.5% buffer, taken up again in 200 μL of water and incubated for 20 min at 60°C, then washed again four times with 200 μL of PBS tween. Finally the balls are taken up again with 25 μL of water and a PCR is performed following the protocol described in example 5. Two reaction controls are performed one with 25 μL of pure water and the other with 2.5 μL of prepared and washed balls under the same conditions as the biological samples, and taken up again in 25 μL of water.
The result:
The PCR products are then quantified by Picogreen (P-7589; Molecular Probes, Eugene, OR, USA) according to the protocol described by the supplier. This method is based on the use of a molecule (Picogreen) which has the property of becoming fluorescent only when it is positioned inside a DNA molecule (by interscaling between bases). Due to the very specific nature of this intercalation, and because the fluorescent signal produced is directly proportional to the amount of DNA present in the medium, it is possible to measure in a very precise way the concentration of nucleic acid in a sample. The signal is then expressed in units of fluorescent (relative r). Analysis of gel PCR results shows the presence of only one band specific to the expected size in samples made from para-Bio-EG3-PDAM-labelled genomic DNA. The bands are not detected when PCR was performed from unlabelled genomic DNA. A quantification of DNA by Picogreen allows for confirmation of DNA production from genomic DNA captured on beads. - What? 21 : Quantification de l'ADN produit par PCR, à partir d'un lysat bactérien, capturé et purifié par le -Bio-EG3-PDAM
Témoins non Marqués 51
ADN marqués 170
Bruit de fond 20
A DNA chip analysis, following the protocol described in Example 8, confirms the specificity of the amplification as shown in Figure 34 below. - What? Détection spécifique de la cible capturée et purifiée par le -Bio-EG3-PDAM
Echantillon 98 11531 723 16
This example shows that it is possible to prepare a biological sample to amplify the nucleic acid it contains by using a capture technique based on the reactivity of the diazomethyl function on its phosphate groups.
Example 38: Successive amplification of two genes from bacterial DNA captured on solid medium:
This example shows that it is possible to amplify a captured DNA repeatedly and on different targets by reactivating the diazomethyl function on its phosphate groups.
In this case, the nucleic acids, contained in a bacterial lysate and marked by para-Bio-EG3-PDAM, are captured on magnetic balls coated with streptavidine. The use of magnetic balls allows these to be preserved by magnetization during successive washes to remove cellular residues from the reaction medium, which must be removed because they can inhibit PCR amplifications, which will be performed later. These amplifications will take place on two different genes present in genomic DNA, respectively designated as 16S and rpoB. These two genes are then analyzed using DNA chips.
The bacterial DNA is obtained by lysis of cells contained in a culture of Mycobacterium tuberculosis, following the protocol already described in Example 37.
10 μL of lysate are incubated in the presence of 20 μL of para-Bio-EG3-PDAM for 30 minutes at 60°C. At the same time, 10 μL of lysate are incubated in 20 μL of pure water (Sigma) under the same conditions.
The reaction medium is then purified on a QIAquick column (Nucleotide Removal Kit, Qiagen, Hilden, Germany).
The marked DNA fragments are then captured on Dynal magnetic balls, which are prepared following the following protocol: Ninety (90) μL of Dynal balls are washed twice with 200 μL of pure Free water (Sigma), then taken up by 200 μL of PEG buffer (0.1 M Na PO4, pH 7.5 M NaCl; 0.65 % Tween 20 ; 0.14 mL Salmon sperm DNA (Gibco) ; 2 % PEG 4000) and incubated 30 min at 37°C. They are then washed twice with 200 μL of PBS 1X Tween 20 0.5% buffer, then finally taken up by 90 μL of the same buffer.
Incubate 10 μL of the marked or unmarked DNA elulate for 5 min at room temperature with 40 μL of PEG buffer and 2,5 μL of the magnetic bead preparation described above.
The balls are then washed three times with 200 μL of PBS 1 X tween 0.5 % buffer, taken up again in 200 μL of water and incubated for 20 min at 60°C, then washed again four times with 200 μL of PBS tween. Finally the balls are taken up again with 25 μL of water and a PCR is performed following the protocol described in example 5. Two reaction controls are performed, one with 25 μL of pure water (Sigma) and the other with 2.5 μL of prepared balls washed in the same conditions as the biological samples, and taken up again, in 25 μL of water.
After amplification, the reaction medium is collected, and the beads are separated and washed with 150 μL of PBS 1 X Tween 0.5%, then resuspended in 25 μL of pure water (Sigma).
Control amplifications from uncaptured genomic DNA are performed in parallel on both amplification systems (rpoB and 16S).
The result:
The resulting PCR products are then analysed by DNA chips following the protocol described in Example 8. - What? Analyse sur puces à ADN des amplicons ADN issus des PCR amplifiées successivement ou non
PCR témoin 16S 16S 96 4662 387 12
Résultats sur séquence rpoB RpoB 23 237 358 1
PCR témoin rpoB RpoB 98 5438 397 14
Résultats sur séquence 16S 16S 17 183 391 1
PCR 16S sur billes 16S 98 2726 534 5
Résultats sur séquence rpoB RpoB 8 161 480 <1
PCR rpoB sur billes lavées RpoB 97 3205 349 9
Résultats sur séquence 16S 16S 14 84 358 <1
Analysis of gel PCR results shows the presence of only one band specific to the expected size in samples taken from para-Bio-EG3-PDAM-labelled genomic DNA. The bands are not detected when PCR has been performed from unlabelled genomic DNA. A DNA chip analysis, as shown in Table 23 above, confirms the specificity of the two successive amplifications and thus the ability to perform a successive amplification of several genes from immobilized DNA on a solid medium, thus avoiding the development of multiplex systems, which often significantly reduce the sensitivity and efficiency of nucleic acid amplifications.
Example 39: Capture and amplification of DNA on a nylon membrane with diazomethyl groups:
A nylon membrane activated to carry diazomethyl groups was used to capture bacterial DNA, with the aim of amplifying it by PCR.
Example 39.1: Modification of the Biodyne C filter: The following is a summary diagram:
Compound 68:
The 3'-aminoacetophenone (14.5 g, 107 mmol) is dissolved in 50 ml of anhydrous DMF. Succinic anhydride (10.7 g, 107 mmol) is added and left to agitate, argon and room temperature. After 6 h, the solution is concentrated under vacuum and 50 ml of methanol is added. The resulting precipitate is filtered and washed with methanol and ether.
The frequency range of the measurement is defined as the frequency range of the measurement.
Compound 69:
After 20 minutes, 20 ml (94.6 mmol) of 4,7,10-trioxatridecanediamine (EG3) is slowly added. After 3 hours of reaction at room temperature, the DMF is evaporated and the residue is taken up again in 100 ml of CH2Cl2. Extractions are made with saturated NaHCO3 and H2O, after which the organic phase is dried with anhydrous Na2SO4 and the evaporated solvent. Thus, 4.34 g (46 g) of the product 69% is obtained.
The following two types of data are used: RMN 1H (200 MHz, DMSO-d6): d= 1,59 (m, 2H); 1,87 (m, 2H); 2,16 (s, 3H); 2,40 (m, 2H); 2,55 (m, 2H); 3,08 (m, 2H); 3,45 (m, 16H); 7,30 (t, 1H); 7,42 (d, 1H); 7,70 (d, 1H); 7,83 (t, 1H); 7,97 (s, 1H); 10,00 (s, 1H).
Compound 91:
A 4 cm2 rectangle is cut on a Biodyne C filter sheet (reference 60314 ; Pall Gelman Laboratory ; Ann Arbor ; Michigan ; USA), introduced into a vial and brought into contact with 0.97 g (6 mmol) of carbonyldiimidazole (CDI) in 3 ml of anhydrous DMF, on ice, under argon and under strong agitation. After 20 min, the solution is removed and the filter washed with DMF. An amount of 0.53 g of product 68 (1 mmol) in 3 ml of anhydrous DMF is then added, and the reaction is carried out overnight at room temperature. The solution is then removed and rinsed under the filter with ethanol, vacuumed and kept dry for argon.
Compound 92:
The modified filter 91 is placed in a solution of 97 ml hydrazine hydrate (2 mmol) in 4 ml of absolute ethanol. The solution is allowed to reflux for 5 h. After cooling, the filter is washed with H2O, ethanol and ether, vacuum dried and placed under argon. Then 4 ml of anhydrous DMF and 86 mg of MnO2 (1 mmol) are added, and reacted under strong agitation. After 20 min, the solution is discarded, and the filter is rinsed with DMF and ether. The modified diazomethyl filter 92 is stored under argon, at -19 to -31°C.
Example 39.2: Biological tests:
The activated membrane is cut into small fragments of 2 mm2 which are incubated for 30 minutes at room temperature in 25 μl of Mycobacterium tuberculosis bacterial lysate prepared by mechanical lysis using the same technique and final concentration as in example 37 and 375 μl of pure water (Sigma).
The membrane is then placed at 65°C for 60 min in 100 mL of washing buffer (5% Formamide (Sigma), 1X SSPE (Perbio), 0.01% Triton X-100) to remove the non-specific nucleic acids adsorbed on the membrane and the membrane is then stored in 1 mL of pure water before amplification.
The PCR shall be performed as described in paragraph 5.1 with a sufficient quantity of pure water to complete the reaction volume.
In parallel, controls are carried out using the same process with membranes which cannot covalently bind nucleic acids: Unmodified Biodyne C Membrane (Membrane A),Membrane Biodyne chemically modified following the process described but not treated with anhydrous DMF and MnO2; this test allows the behaviour of the membrane to be verified in the absence of diazomethyl groups (Membrane B), andMembrane Biodyne C unmodified but treated for 20 min under strong agitation by anhydrous DMF and MnO2, this test allows the verification that the latter step does not alter the adsorption of DNA on the membrane (Membrane C).
To check that the membrane treatment does not inhibit PCR, another fragment of the A, B, C membranes is amplified simultaneously with 25 μl of bacterial lysate.
The PCR products are then quantified by Picogreen according to the protocol described by the supplier.
The result:
Quantification de l'ADN obtenu par PCR à partir d'ADN de lysat bactérien capturé sur support solide
Membrane modifiée 111
Membrane non modifiée (A) 18
Membrane non modifiée, co-amplifiée avec 25 µL de lysat bactérien (A') 260
Membrane modifiée non activée (B) 26
Membrane modifiée non activée, co-amplifiée avec 25 µL de lysat bactérien (B') 264
Membrane non modifiée ayant subit une activation (C) 21
Membrane non modifiée ayant subit une activation, co-amplifiée avec 25 µL de lysat bactérien (C') 268
These results in Table 24 indicate that it is possible to capture covalently on a solid medium, nucleic acids from lysate, by means of diazomethyl chemistry. The amplification observed is not due to non-specific adsorption of DNA on the membrane.
To check the nature of the amplified product on the membrane, the amplification product was analysed by DNA chip passing, following the protocol described above.

Claims (28)

  1. Method for labelling and fragmenting a single-stranded or double-stranded deoxyribonucleic acid (DNA), comprising:
    - fragmenting the DNA by means of the creation of at least one abasic site on said DNA,
    - attaching a label to at least one of the fragments by means of a labelling reagent, said reagent coupling covalently and predominantly to at least one phosphate of said fragment, the fragmentation and the labelling being carried out in one step.
  2. Method for the provision of probes for detecting a target nucleic acid, comprising the labelling and the fragmentation of a single-stranded or double-stranded deoxyribonucleic acid (DNA), comprising the following steps:
    - fragmenting the DNA by means of the creation of at least one abasic site on said DNA,
    - attaching a label to at least one of the fragments by means of a labelling reagent, said reagent coupling covalently and predominantly to at least one phosphate of said fragment.
  3. Method according to Claim 2, characterized in that the fragmentation and the labelling are carried out in two steps.
  4. Method according to Claim 2, characterized in that the fragmentation and the labelling are carried out in one step.
  5. Method according to any one of Claims 1 to 4, characterized in that the abasic site is generated by the action of an acidic aqueous medium.
  6. Method according to Claim 5, characterized in that the pH of the acidic medium is less than 5, preferably less than 4.
  7. Method according to Claim 6, characterized in that the acidic aqueous medium is a sodium formate buffer having a pH of approximately 3.
  8. Method according to any one of Claims 1 to 7, characterized in that the DNA contains at least one modified base capable of generating an abasic site.
  9. Method according to Claim 8, characterized in that the modified base capable of generating an abasic site is chosen from 8-bromopurine derivatives.
  10. Method according to any one of Claims 1 to 4, characterized in that the abasic site is generated by an alkylating agent or an oxidizing agent.
  11. Method according to any one of Claims 1 to 10, characterized in that the labelling reagent comprises, as reactive function, a unit chosen from the following compounds: diazomethyl; alkyl halide; nitrosourea; spirocyclopropane; aziridine; epoxide; trifluorosulfonates.
  12. Method according to Claim 11, characterized in that the labelling reagent is 5-(bromomethyl)-fluorescein.
  13. Method according to Claim 11, characterized in that the labelling reagent is chosen from the compounds of formula (1): in which:
    • R1 represents H or an alkyl, substituted alkyl, aryl or substituted aryl group, and
    • Z comprises a detectable label.
  14. Method according to Claim 13, characterized in that Z is:
  15. Method according to Claim 14, characterized in that the labelling reagent is chosen from the compounds of formula (2): in which
    • R1 represents H or an alkyl, aryl or substituted aryl group,
    • R2 is a detectable label,
    • L is a linker arm containing a linear chain of at least two covalent bonds and n is equal to 0 or 1, and
    • Z is chosen from: in which:
    ■ R3 and R4 represent, independently of one anqther: H, NO2, Cl, Br, F, I, OR, SR, NR2, R, NHCOR, CONHR or COOR with R = alkyl or aryl, and
    ■ -Y-X- represents -CONH-, -NHCO-, -CH2O-, -CH2S-.
  16. Method according to Claim 15, characterized in that the labelling agent has the formula (3): in which:
    • R1 represents H or an alkyl, aryl or substituted aryl group,
    • R2 represents a detectable label,
    • L is a linker arm containing a linear chain of at least 2 covalent bonds and n is an integer equal to 0 or 1,
    • R3 and R4 represent, independently of one another: H, NO2, Cl, Br, F, I, OR, SR, NR2, R, NHCOR, CONHR or COOR with R = alkyl or aryl, and
    • -Y-X- represents -CONH-, -NHCO-, -CH2O- or -CH2S-.
  17. Method according to either one of Claims 15 and 16, characterized in that R2 is a fluorescent compound or a hapten.
  18. Method according to either one of Claims 16 and 17, characterized in that said labelling reagent is soluble in a water-miscible solvent.
  19. Method for detecting a single-stranded or double-stranded target deoxyribonucleic acid (DNA), comprising the following steps:
    • fragmenting and labelling said DNAs, according to any one of the methods according to Claims 1 to 18,
    • hybridizing the labelled fragments to at least one nucleic acid probe sufficiently specific for the target nucleic acid, and
    • detecting the hybrid formed, by means of the label.
  20. Method for detecting a double-stranded target deoxyribonucleic acid (DNA), according to Claim 19, also comprising a denaturation step after the fragmentation and the labelling.
  21. Method according to Claim 20, characterized in that the fragmentation, the labelling and the denaturation are carried out in a single step.
  22. Method for detecting a target nucleic acid, comprising the following steps:
    • enzymatically amplifying the target nucleic acid so as to generate a multitude of double-stranded DNA amplicons,
    • fragmenting and labelling said DNA amplicons, according to any one of the methods according to Claims 1 to 18,
    • hybridizing the labelled fragments to at least one nucleic acid probe sufficiently specific for the target nucleic acid, and
    • detecting the hybrid formed, by means of the label.
  23. Method according to Claim 22, also comprising a denaturation step after the fragmentation and labelling step.
  24. Method according to Claim 23, characterized in that the fragmentation, the labelling and the denaturation are carried out in a single step.
  25. Method for demonstrating a polymorphism distributed in possibly predetermined positions of a target nucleic acid, by means of the presence of a plurality of deletions and/or insertions and/or mutations in the sequence of said target nucleic acid compared with a "reference" sequence, comprising the following steps:
    • providing a target DNA containing the entire polymorphism to be studied, said DNA being optionally generated by means of an enzymatic amplification technique,
    • fragmenting and labelling said DNA by means of a method according to any one of Claims 1 to 18,
    • hybridizing said fragments to a plurality of nucleic acid probes referred to as capture probes, the plurality of capture probes being attached to a solid support and the plurality of capture probes covering, in its entirety, at least the polymorphism to be studied,
    • detecting the hybrids formed between the labelled fragments and at least some of the nucleic acid probes, by means of the label, and deducing therefrom the polymorphism of the target DNA.
  26. Method according to Claim 25, also comprising a denaturation step after the fragmentation and labelling step.
  27. Method according to Claim 26, characterized in that the fragmentation, the labelling and the denaturation are carried out in a single step.
  28. Method according to any one of Claims 25 to 27, characterized in that the solid support comprises at least ten (10) nucleic acid probes of different sequences, advantageously at least four hundred (400), preferably at least a thousand (1000).
HK04105111.9A 2001-05-04 2002-05-03 Method for labelling and fragmenting dna HK1062033B (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
FR0106039A FR2824335A1 (en) 2001-05-04 2001-05-04 DNA MARKING AND FRAGMENTATION PROCESS
FR01/06039 2001-05-04
PCT/FR2002/001542 WO2002090584A2 (en) 2001-05-04 2002-05-03 Method for labelling and fragmenting dna

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Publication Number Publication Date
HK1062033A1 HK1062033A1 (en) 2004-10-15
HK1062033B true HK1062033B (en) 2007-04-27

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