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WO2004044242A1 - Procede de detection de polymorphisme - Google Patents

Procede de detection de polymorphisme Download PDF

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Publication number
WO2004044242A1
WO2004044242A1 PCT/GB2003/004957 GB0304957W WO2004044242A1 WO 2004044242 A1 WO2004044242 A1 WO 2004044242A1 GB 0304957 W GB0304957 W GB 0304957W WO 2004044242 A1 WO2004044242 A1 WO 2004044242A1
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WIPO (PCT)
Prior art keywords
oligonucleotide
polymoφhism
polynucleotide
polynucleotide molecule
hybridised
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Ceased
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PCT/GB2003/004957
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English (en)
Inventor
John Nicholas Housby
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Individual
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Priority claimed from GB0226534A external-priority patent/GB0226534D0/en
Application filed by Individual filed Critical Individual
Priority to AU2003283575A priority Critical patent/AU2003283575A1/en
Publication of WO2004044242A1 publication Critical patent/WO2004044242A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • the present invention is concerned with a new method for analysing polymorphisms, in particular single nucleotide polymorphisms (SNP's), that is not dependent upon the PCR amplification and requires only small quantities of chromosomal DNA spotted onto a microarray surface.
  • SNP's single nucleotide polymorphisms
  • the invention is also concerned with the production of a kit that contains the reagents and materials necessary to use the technology.
  • Polymorphisms are changes in the sequence of bases in the DNA sequence of the human, or other, genome. Sometimes the base change leads to a change in the translated amino acid residue that it codes for, the protein sequence, which in turn may alter the function of the protein. The majority of polymorphisms are 'silent', they do not alter the protein sequence but they may affect the expression of the gene. Polymorphisms may reside in promoter regions where their effect on gene expression may be monitored, or in intronic sequence where their function may not be known, or in terminal gene sequences at the start or end of the gene where their function may be regulatory.
  • SNP's single nucleotide polymorphisms
  • GBA Genetic Bit Analysis
  • OBA Oligonucleotide Ligation Assay
  • ASO allele specific oligonucleotide
  • a reporter oligonucleotide is designed to hybridise downstream of the SNP with a complementary SNP base at its 3' end.
  • Taq DNA ligase is then used to discriminate this SNP base as the ligase will not allow mismatched bases at the SNP site to be ligated.
  • DNA is separated by gel electrophoresis and transferred onto nitrocellulose paper, prior to assaying with radiolabelled probes.
  • LDR Ligase Detection Reaction
  • LDR Ligase Detection Reaction
  • several rounds of ligation are applied to the reaction in a way similar to PCR amplification.
  • the oligonucleotides used to detect the SNP are hybridised and ligated by DNA ligase. This is followed by denaturation of the hybridised duplex and a further round(s) of hybridisation and denaturation are performed.
  • the products of ligation are 'captured' onto a microarray of complementary DNA sequence.
  • the 'captured' ligation products are detected on the microarray by fluorescence based methods.
  • WO 02/053778 discloses a method for identification of nucleic acids by using a PCR template and using a labelled oligonucleotide to detect, for example, SNPs. This technique uses complex PCR to amplify the DNA, prior to assaying for the SNP. This is a variation of the oligonucleotide ligation assay described above.
  • WO 00/63437 discloses assays for SNPs, using a capture probe which is an oligonuceotide, attached to a microsphere on a substrate.
  • the capture probe captures nucleotides comprising the SNP or products derived from the nucleotides.
  • the nucleotides contained in the SNP are indicated as being the products of a PCR reaction.
  • WO 00/56927 discloses a method of detecting SNPs and involves using a PCR reaction followed by a ligase detection reaction.
  • the products of the ligase detection reaction are either detected by hybridising the ligated oligonucleotide probes to a solid support with an array of immobilised capture oligonucleotides, at least some of which are complimentary to addressable array-specific portions on the oligonucleotides.
  • the SNPs are detected by means of capillary electrophoresis or gel electrophoresis and fluorescent identification.
  • WO 01/85987 discloses a method for identifying or detecting a nucleotide repeat region having a particular sequence in a nucleic acid molecule, for example in the detection of nucleotide length polymorphisms.
  • This uses a variant of ligase detection reaction for amplifying the nucleic acid molecules to be assayed, in a similar manner to PCR.
  • the amplified nucleic acid is itself labelled and biotinylated. This biotinylated, labelled nucleic acid molecule is then attached to a solid support and detected.
  • EP 1130113 discloses a ligation-dependent amplification assay, but involves the amplification of the ligation products. The amplified ligation products are then detected.
  • US 5,866,337 discloses a method for detecting mutations in nucleic acid molecules by contacting the nucleic acid molecules with a probe.
  • the single probe comprises two covalently linked nucleic acid segments that are hybridised with the nucleic acid molelcule.
  • the mixture is then contacted with a ligase so that the two hybridised probe segments ligate and bind the nucleic acid molecule if the nucleic acid molecule contains a mutation. This produces a catenation product.
  • EP 0185494A discloses an assay for the diagnosis of genetic abnormalities. This uses two oligonucleotide probes, one of which is labelled. Two probes bind either side of the genetic abnormality. The amount of label, such as radioactive 32 P, released upon raising the temperature to melt the probe-DNA duplex, is measured. This gives an indication of the presence of a genetic abnormality, but at low sensitivity.
  • the assay does not allow the possibility of multiplexing, that is looking for a number of separate abnormalities in the same sample of DNA at the same time.
  • Landegern U., et al. discloses a method of identifying a gene comprising contacting two oligonucleotides in solution with a DNA molecule.
  • the DNA molecule is then denatured, the two oligonucleotides allowed to anneal and ligate if the target sequence of interest is present, the target-oligonucleotide sequences denatured and any ligated oligonucleotides formed then allowed to bind to a biotin binding support on a bead.
  • This system used a radioactive label and did not allow the multiple targeting of different DNA sequences at the same time.
  • WO 01/73122 discloses a method for determination of SNPs using a bio-electronic microchip. This uses oligonucleotides or template probes which are bound onto a support by electrostatic binding. The method requires amplification to achieve the required assay and it is oligonucleotides, not chromosome DNA, that is bound to the microchip.
  • this invention can utilise the patients' un-amplified polynucleotides, such as DNA, directly on a microarray surface. It is expected that the method will reduce the cost of genotyping.
  • the invention draws on previous patented technologies but differs in the overall combination and approach to genotyping.
  • the invention will impact on the industry by increasing the throughput of genotyping and lowering the cost of reagents and the cost of personnel.
  • the invention will not need expensive equipment like mass spectrometry (Third Wave) or fibre optics (Illumina) but may be of advantage to those companies that already possess robotic spotting technology.
  • the technique also reduces the need for gel electrophoresis of samples prior to assaying. Furthermore, the technique allows a plurality of different plymorphisms to be detected at the same time (so-called "multiplexing").
  • the invention provides a method for determining the presence or absence of a first polymo ⁇ hism in a polynucleotide molecule comprising the steps of:
  • the two oligonucleotides will not form a circular catenation product.
  • the substrate is a planar non-porous solid support having a first surface on which the polynucleotides are attached. This may be rigid or semi-rigid. In many embodiments the surface will be substantially flat, although in some embodiments it may be desirable to have wells, raised regions, etched trenches or the like.
  • the substrate is a microtitre plate or microchip.
  • the plurality of polynucleotide molecules are preferably spotted onto the surface of the substrate to produce a predefined pattern of spots, each spot containing a plurality of polynucleotide molecules.
  • the substrate surface may eventually comprise one or more rows or columns of discrete areas of polynucleotide molecules attached to the surface.
  • the surface of the substrate may just comprise polynucleotide molecules, such as chromosomal, genomic DNA, from one source.
  • polynucleotide molecules from two or more different sources such as different patients or different tissue samples, may be attached to the substrate; each source of polynucleotide molecules being placed on a different, predefined, part of the substrate surface.
  • Each predefined region may be less than 1 mm, less than 100 micrometers, less than 20 micrometers, especially 10 micrometers in diameter. They may be placed in a substantially regular pattern.
  • Preferred density for the predefined regions are 1,000 - 10,000 cm 2 .
  • the polymorphism may be a single nucleotide polymo ⁇ hism. Alternatively, it may be a polymo ⁇ hism covering two or more base pairs in the polynucleotide molecule, for example caused by deletion, addition or substitution of bases.
  • high stringency conditions we mean that the conditions under which the second oligonucleotide and first oligonucleotide are allowed to hybridise are such that the second oligonucleotide will bind specifically to a predetermined polymo ⁇ hism sequence and not to a second, different, polynucleotide sequence.
  • the polymo ⁇ hism to be detected may be an adenine nucleotide in a predetermined position of the polynucleotide molecule.
  • a probe containing a thymidine residue in the oligonucleotide will allow the oligonucleotide to hybridise to the polymo ⁇ hism.
  • the adenine is not present at the polymo ⁇ hism, but is instead replaced, for example, by a cytosine residue
  • a probe containing a thymidine residue will not hydrogen bond with the cytosine residue and will not hybridise to the polynucleotide. This means that the first oligonucleotide and the second oligonucleotide will not be hybridised to the polynucleotide molecule and will not be ligated.
  • the ligated first and second oligonucleotide are detected in situ, that is hybridised to the polynucleotide molecule. Unbound first oligonucleotide and second oligonucleotide may be washed away from the polynucleotide attached to the substrate.
  • the first oligonucleotide is preferably longer than the second oligonucleotide.
  • the first oligonucleotide preferably binds to a predetermined locus on the polynucleotide. This oligonucleotide preferably hybridises adjacent, but upstream, 5' to the polymo ⁇ hism of interest.
  • the first oligonucleotide is less than 500, less than 400, less than 300, less than 200, less than 150, preferably less than 100 bases in length.
  • the second oligonucleotide is preferably less than 30, less than 25, less than 20, most preferably less than 15 nucleotides in length. It is designed to specifically hybridise, that is to correctly base pair with the predetermined nucleotide sequence.
  • the stringency conditions may be varied to allow the discrimination between bound and non-bound second oligonucleotide by, for example, varying the temperature of the mixture. Increasing the temperature increases the stringency. The second oligonucleotide which correctly base pairs with the predetermined polymo ⁇ hism sequence will remain hybridised under higher temperatures than mismatched oligonucleotides.
  • the salt concentrations of the reaction mixture may be varied. Lowering the salt concentration results in a higher stringency for the reaction mixture.
  • Typical high stringency conditions are: 0.5% SSC, 0.1% SDS, or 1.5 mM MgCl 2 , 52°C
  • the polynucleotide molecule may be DNA or RNA. This includes genomic and mitochondrial DNA or mRNA.
  • the polynucleotide molecules are genomic DNA that may have fragmented, for example by shearing, prior to attaching to the substrate.
  • the DNA is not amplified by multiple amplication reactions such as using PCR or other amplication methods used in the art. This reduces the time needed to carry out the assay. If amplification is carried out, preferably only one round of amplification is carried out to produce one copy of the polynucleotide..
  • mRNA may be converted into cDNA by, for example, RT-PCR. This latter technique allows the production of labelled cDNA to be produced and the amount of any polymo ⁇ hism produced to be calculated by the amount of labelled ligated first and second oligonucleotides to be determined.
  • the 5' end of the cDNA may be aminated by methods known in the art, to allow the cDNA to be attached to the substrate, prior to looking for the presence or absence of the polymo ⁇ hism.
  • the DNA is denatured prior to hybridisation with the oligonucleotides.
  • the oligonucleotides are DNA, RNA or PNA (peptide nucleic acid) or analogues thereof.
  • DNA or RNA analogues may be made, for example, by replacing the sugar-phosphate backbone with an analogue, such as a sugar-sulphur backbone.
  • PNA is a nucleic acid analogue with a peptide rather than a sugar-phosphate backbone.
  • PNAs have high thermal stability and also exhibit greater sensitivity to mismatches, thus being ideally suited for probing, such as SNP's.
  • smaller probes can be used, which further increases the specificity of the oligonucleotide.
  • PNA probes are reviewed, for example, in The Principle, Vol. 12, page 17 (1998) and are discussed in the paper by Ni, P.E., et al., Science, Vol. 254, pages 1497-1500 (1991).
  • the first and second oligonucleotide are ligated by means of a ligase, such as a thermostable ligase.
  • a ligase such as a thermostable ligase.
  • Ligases attach the 3' end of one oligonucleotide to an adjacent 5' end of an adjacent oligonucleotide.
  • ligases are known in the art.
  • the advantage of using a thermostable ligase is that high temperatures can be used to produce the high stringency required to discriminate between correctly base matched second oligonucleotide and incorrectly matched oligonucleotides.
  • the method of the invention may additionally comprise the step of:
  • detecting ligated first and third oligonucleotide (iii) detecting ligated first and third oligonucleotide.
  • the use of the third oligonucleotide allows the discrimination between, for example, two different SNP's.
  • the third oligonucleotide and the second oligonucleotide may be added to the polynucleotide molecule at the same time. If, for example, the second and third oligonucleotides are labelled with different labels, this allows the rapid identification of the polymo ⁇ hism present in the polynucleotide molecule.
  • the detection of the ligated first oligonucleotide to the second or third oligonucleotides may be carried out using methods known in the art, such as using a radio-label, using horseradish peroxidase, using antibody to detect the ligated oligonucleotides or using biotin/avidin, or indeed alkaline phosphatase.
  • the label is a fluorescent or visible label.
  • fluorescent labels are well known in the art. Examples include Cy3 or Cy5. Choosing different labels for different oligonucleotides would allow the discrimination between, for example, the presence of ligated second or third oligonucleotides.
  • the use of fluorescently labelled probes also means that the technique can easily be used using existing photo-detection equipment of the type commonly used in laboratories.
  • the second or third oligonucleotides which are labelled. This means that if the second or third oligonucleotides do not bind they can simply be washed away, thus removing the label from the system.
  • the labelling system used is a dendrimer-based system.
  • This method of labelling uses a dendrimer to attach 200 or more fluorescent labels allowing the detection of a small number of target molecules.
  • dendrimer technology is produced, for example, by Genisphere, Bala, Cynwyd, PA, USA.
  • Quantum Dots may be obtained, for example, from Quantum Dot Co ⁇ oration, Hayward, CA, USA.
  • the polymo ⁇ hism uses polynucleotide molecules which have been sheared, for example by physical shearing or, alternatively, by the use of a nuclease such as a restriction endonuclease.
  • the polynucleotide samples may be dotted onto a micro-array plate or microchip. Separate dots may then be challenged with different sets of first and second oligonucleotides, each set of oligonucleotides directed to a different predetermined polymo ⁇ hism.
  • nano particles which allow the use of over 100 different colours which are detectable by the same ultraviolet light source at the same wavelength, many tens, or even hundreds, of different polymo ⁇ hisms may be detected at the same time. For example, at least 50 genotypes could be determined from one sample on a microchip surface.
  • the assay of the invention may be extended. Accordingly, the invention also provides a method of identifying the presence or absence of two or more linked polymo ⁇ hism sites in a polynucleotide molecule comprising determining the presence or absence of a first polymo ⁇ hism site by the method according to the invention;
  • a fourth oligonucleotide capable of specifically hybridising to a third polymo ⁇ hism sequence corresponding to a predetermined polymo ⁇ hism sequence of the second polymo ⁇ hism site;
  • an alternative oligonucleotide (a fifth oligonucleotide) which is capable of specifically hybridising to a different polymo ⁇ hism sequence at the second polymo ⁇ hism site may be used instead of or at the same time as the fourth oligonucleotide.
  • the use of different labels allows the detection of, for example, the presence of the first polymo ⁇ hism site and not the second polymo ⁇ hism site, or alternatively, or indeed additionally, the specific sequences at the polymo ⁇ hism sites.
  • the method may be used to assay for several different polymo ⁇ hisms at the same time.
  • different oligonuclotides may be used to detect different polymo ⁇ hisms within each sample of DNA, to detect the presence or absence of several SNPs or other variations associated with different phenotypic traits.
  • the different oligonucleotides may be detected using different labels.
  • the polymo ⁇ hisms need not be linked on the same polynucleotide molecule .
  • a further aspect of the invention provides a method comprising determining the presence or absence of two or more polymo ⁇ hisms.
  • the invention also provides a substrate comprising:
  • this is prior to ligation of the molecules.
  • the polynucleotide molecule is preferably genomic DNA, mitochondrial DNA or mRNA.
  • the substrate may additionally comprise a third oligonucleotide specifically hybridised to a second predetermined polymo ⁇ hism site on the polynucleotide molecule.
  • the oligonucleotides and substrate may be as defined above.
  • a kit for identifying the presence or absence of a first polymo ⁇ hism in a polymo ⁇ hism sequence comprising:
  • (iii) means to ligate the first and second oligonucleotides.
  • the kit is preferably for use with the method of the invention.
  • the kit also comprises means to ligate the first and second oligonucleotide, such as a thermostable ligase.
  • the oligonucleotides may be labelled by labels as described above.
  • the kit may additionally comprise a third oligonucleotide capable of specifically hybridising to a second predetermined polymo ⁇ hism on the polynucleotide molecule.
  • the kit is used to determine the presence or absence of a first polymo ⁇ hism in a polynucleotide molecule.
  • the kit may also be used to determine the presence of a second polymo ⁇ hism in a polynucleotide molecule.
  • the components of the kit are preferably as defined above.
  • Figure 1 shows a schematic diagram representing the method of the invention.
  • Figure la shows a piece of sheared genomic DNA (10) which has been attached to a substrate (12) such as a microchip surface by methods known in the art.
  • a first oligonucleotide (16) is introduced to the genomic DNA (10).
  • the first oligonucleotide is designed to hybridise adjacent to a polymo ⁇ hism site (represented as X and 14). This identifies the locus of the polymo ⁇ hism.
  • the second and third oligonucleotides (18 and 20) are introduced.
  • the second and third oligonucleotides are designed to hybridise to different sequences at the polymo ⁇ hism site. They are labelled with different labels.
  • the second oligonucleotide (18) hybridises to the genomic DNA (10) and the polymo ⁇ hism site (14).
  • the conditions used are such that they are under high enough stringency to specifically allow the binding of the second oligonucleotide (18) but not the third oligonucleotide (20) owing to the difference in the base matching between the polymo ⁇ hism site and the oligonucleotides.
  • the inbound oligonucleotide (20) is then washed away.
  • the labelled oligonucleotide is then detected by simple detection means.
  • the isolation of chromosomal DNA may be carried out using the methods of Sambrook, et al. (Molecular Cloning, a Laboratory Manual (1987), Coldspring Harbour Laboratory Press). Shearing of DNA may be carried out using physical shearing methods or, for example, enzymes such as restriction endonucleases. For shearing, methods well known in the art may be used, such as shown, for example, in Sambrook, et al. (Supra).
  • cDNA is attached to the substrate, instead of chromosomal DNA, then cDNA is produced using mRNA as a template. This may use random hexamers as oligonucleotide primers to prime the reaction. For gene expression studies, the random hexamer oligonucleotides may be aminated prior to the priming reaction. This would enable the hexamers to be immobilised on the arrays using chemistry known in the art. mRNA may then be converted into cDNA using methods well known in the art.
  • a substrate such as a glass substrate
  • a substrate such as a glass slide
  • is cleaned for example with a solution of sodium hydroxide in ethanol and distilled water.
  • the cleaning solution is then removed and the slides washed with distilled water.
  • a solution of poly-L-lysine is then made up.
  • such solutions typically contain 70 mL poly-L-lysine, 70 mL phosphate buffered saline and 560 mL water.
  • Phosphate buffered saline typically contains 8 grams of sodium chloride, 0.2 grams of potassium chloride, 1.44 grams of sodium phosphate (dibasic anhydrous) and 0.24 grams of monobasic potassium phosphate per litre of distilled water.
  • the glass slides are soaked in the polylysine solution for 15 mins. - 1 hour, prior to rinsing with distilled water.
  • the prepared slides are then dried. Solutions containing DNA are then dropped onto the slide to form an array of polynucleotide molecules, and dried to attach the DNA to the substrate.
  • DNA may be attached via charge interactions to GAPS II glass slides which are available from Corning.
  • Polynucleotides may also be attached to substrates via isothiocyanate chemistry. This method is described, for example, in the article by Beier and Hoheisel (1999), Nucleic Acids Research, Vol. 27 (9), pages 1970-7. Briefly, glass slides are silanised by immersion in 10% Na OH for approximately 18 hours. The slides are then washed in distilled water and then successive washes in 1% HCl/H 2 0/methanol. The slides are then sonicated in 3% aminopropyltrimethoxysilane, followed by washing in distilled water and methanol. The slides are then dried in a stream of nitrogen, prior to baking for 110°C for 15 mins. For amidated oligonucleotides, the slides may be activated by phenylenediisothiocyanate (PDITC).
  • PDITC phenylenediisothiocyanate
  • Mercaptosilane modified glass surfaces may be used. This method has been described, for example, by Rogers, et al. (1999), Anal. Biochem., Vol. 266 (1), pages 23-30. Briefly, glass slides are cleaned by soaking in 25% ammonium hydroxide for at least 16 hours, followed by rinsing in distilled water for 10 mins. The glass slides are silanised by immersing in a 1% solution of 3-mercaptopropyl-trimethoxysilane (MPTS), 95% ethanol, 16 mM acetic acid (pH 4.5), for 30 mins. The plates are then rinsed in 95% ethanol/16 mM acetic acid (pH 4.5) and dried in a vacuum oven for 2 hours at 150°C.
  • MPTS 3-mercaptopropyl-trimethoxysilane
  • MPTS 3-mercaptopropyl-trimethoxysilane
  • the plates are then rinsed in 95% ethanol/16 mM acetic acid (pH
  • Target DNA is then immobilised via a thiol/disulphide exchange reaction.
  • Hybridisation of the oligonucleotide probes to the immobilised DNA may be performed according to the prior art (Housby and Southern (1998), Nucleic Acids Research, Vol. 26, pages 4259-66). Briefly, a solution of ligation buffer for Thermus thermophilus (Tth), DNA ligase (Advanced Biotechnologies Ltd.) and 2 oligonucleotides that define the SNP genotype is pipeted onto a glass slide containing samples of DNA attached to it. The slide and solution are incubated at 46°C in a hybridisation chamber or under coverslip. By reducing the temperature in a controlled manner, for example 1°C per second, the oligonucleotides will hybridise specifically to their complementary DNA target.
  • Incubation of the reaction can be from 4 hours to overnight followed by washing in ligation buffer (1 times) for 10-15 mins. at greater than 10°C of the Tm of the shorter oligonucleotide.

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Abstract

L'invention concerne un procédé permettant de déterminer la présence ou l'absence d'un premier polymorphisme dans une molécule polynucléotidique. Les étapes de ce procédé consistent à (i) immobiliser une pluralité de molécules polynucléotidiques sur une pluralité de zones prédéfinies d'un substrat afin de former un ensemble ordonnée de molécules polynucléotidiques, (ii) à produire une hybridation d'un premier oligonucléotide dans une position adjacente au premier polymorphisme avec au moins une molécule polynucléotidique, (iii) à ajouter un second oligonucléotide, capable de s'hybrider de manière spécifique avec une première séquence nucléotidique présentant un polymorphisme prédéterminé, (iv) à provoquer la ligation d'au moins une partie du second oligonucléotide qui est hybridé avec la molécule polynucléotidique, avec le premier oligonucléotide qui est hybridé avec le polynucléotide dans des conditions de stringence élevée, et (v) à détecter le premier et le second oligonucléotide ayant subi une ligation in situ. Les molécules de polynucléotides sont de préférence de l'ADN génomique, de l'ADN mitochondrial ou de l'ARNm. L'invention concerne en outre des trousses destinées à être utilisées dans le procédé décrit.
PCT/GB2003/004957 2002-11-14 2003-11-14 Procede de detection de polymorphisme Ceased WO2004044242A1 (fr)

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AU2003283575A AU2003283575A1 (en) 2002-11-14 2003-11-14 Polymorphism assay

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GB0226534A GB0226534D0 (en) 2002-11-14 2002-11-14 A quicker method for polymorphism analysis using chromosomal DNA and microarrays than those available currently
GB0226534.6 2002-11-14
GB0306833.5 2003-03-25
GB0306833A GB0306833D0 (en) 2002-11-14 2003-03-25 Polymorphism assay

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EP0246864A2 (fr) * 1986-05-19 1987-11-25 Bio-Rad Laboratories, Inc. Sondes d'hybridation
WO1999060007A2 (fr) * 1998-05-15 1999-11-25 Isis Innovation Limited Reactif et procede
WO2000005412A1 (fr) * 1998-07-24 2000-02-03 Lumigen, Inc. Synthetisation de polynucleotides par ligature d'oligomeres multiples
WO2001049881A2 (fr) * 2000-01-01 2001-07-12 Agrobiogen Gmbh Biotechnologie Matrices d'adn et leur utilisation pour examiner des individus d'une population
WO2001073122A1 (fr) * 2000-03-28 2001-10-04 Nanogen, Inc. Procedes servant a determiner des polymorphismes d'acides nucleiques simples au moyen d'un puce bioelectrique
WO2002003849A2 (fr) * 2000-07-10 2002-01-17 Telechem International, Inc. Technique de genotypage de plusieurs echantillons sur plusieurs loci utilisant des micro-reseaux
US6344316B1 (en) * 1996-01-23 2002-02-05 Affymetrix, Inc. Nucleic acid analysis techniques
US20020086289A1 (en) * 1999-06-15 2002-07-04 Don Straus Genomic profiling: a rapid method for testing a complex biological sample for the presence of many types of organisms

Patent Citations (9)

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