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WO2000071698A2 - Melange pour neutraliser des inhibiteurs d'enzymes - Google Patents

Melange pour neutraliser des inhibiteurs d'enzymes Download PDF

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Publication number
WO2000071698A2
WO2000071698A2 PCT/EP2000/003933 EP0003933W WO0071698A2 WO 2000071698 A2 WO2000071698 A2 WO 2000071698A2 EP 0003933 W EP0003933 W EP 0003933W WO 0071698 A2 WO0071698 A2 WO 0071698A2
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
mixture
mixture according
reaction
blood
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2000/003933
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German (de)
English (en)
Other versions
WO2000071698A3 (fr
Inventor
Christian Reiter
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Connex Gesellschaft zur Optimierung von Forschung und Entwicklung mbH
Original Assignee
Connex Gesellschaft zur Optimierung von Forschung und Entwicklung mbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Connex Gesellschaft zur Optimierung von Forschung und Entwicklung mbH filed Critical Connex Gesellschaft zur Optimierung von Forschung und Entwicklung mbH
Priority to AU47543/00A priority Critical patent/AU4754300A/en
Publication of WO2000071698A2 publication Critical patent/WO2000071698A2/fr
Publication of WO2000071698A3 publication Critical patent/WO2000071698A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/18—Apparatus specially designed for the use of free, immobilized or carrier-bound enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/20—Material Coatings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present invention relates to a mixture of albumin and / or spermidine and at least one antibody, preferably a mixture of albumin, preferably bovine serum albumin, spermidine and an antibody or a solution containing this mixture.
  • This mixture can be used to produce a reaction buffer or to coat reaction vessels. This can neutralize substances that inhibit enzymes used for the manipulation of nucleic acids and are normally present in samples, thereby reducing the efficiency of enzymatically catalyzed nucleic acid modifications, e.g.
  • PCR Polymerase chain reaction
  • nucleic acids for example genomic DNA
  • biological material for example to be able to amplify them by means of PCR
  • digest the sample which, for example, involves heating the sample and enriching and purifying the nucleic acid in order to thereby inhibit the enzymatic modification reaction to destroy or remove.
  • Common purification methods are, for example, ion exchange chromatography and digestion with proteases, for example proteinase K.
  • proteases for example proteinase K.
  • inhibitors of such reactions include hemin, humic acids, heparin, gall salts and certain sugar structures.
  • the cleaning methods described above have the disadvantage that they do not always remove the inhibitors to a sufficient extent, which is then a requires further purification of the nucleic acids.
  • the associated disadvantages include not only the high expenditure of time and money, but also the loss or degradation of the nucleic acid to be manipulated (especially with high molecular weight DNA or RNA).
  • the invention is therefore based on the object of providing means with which inhibitors of enzymes used in the manipulation of nucleic acids can be effectively neutralized.
  • antibody used here refers to both a polyclonal and monoclonal antibody.
  • the mixture can also contain more than one antibody.
  • the albumin used is preferably bovine serum albumin and the antibody used is an antibody of the IgG class.
  • the experiments carried out showed that the antibody has a clear inhibitory effect, regardless of its specificity, which exceeds that of a normal protein, for example BSA.
  • the observed effect (see Example 5) with regard to the individual components (spermidine, BSA, antibody) is not additive, but synergistic. IgG antibodies have proven to be particularly effective.
  • the invention further relates to a solution which contains the mixture according to the invention.
  • Solutions in which the concentration of albumin is 0.4 ⁇ g / ⁇ l, of spermidine 0.2 ⁇ g / ⁇ l and of the antibody 0.2 ⁇ g / ⁇ l are particularly preferred.
  • the mixture according to the invention or the solution containing it is advantageously to be used in the manipulation of nucleic acids contained in biological samples, in particular for use in routine diagnostic applications.
  • biological samples are materials which are known to contain high concentrations of inhibitors, for example blood, blood components, soil samples, tissue homogenates, fixed tissue, lavage, cerebrospinal fluid, stool for the detection of Helicobacter pylori and urine, preferably for the detection of chlamydia.
  • success can be achieved in various fields of medicine in which a diagnosis (based on the problems described above) was not possible at all via PCR, for example when detecting individual tumor cells in
  • the mixture according to the invention can be used in the detection of sepsis pathogens via PCR, which is thereby much faster and also more sensitive than the detection of the pathogens via cultivation.
  • the present invention thus also relates to the use of the mixture according to the invention or of the solution according to the invention for
  • Reaction vessels for neutralizing inhibitors of enzymes used in the manipulation of nucleic acids are Reaction vessels for neutralizing inhibitors of enzymes used in the manipulation of nucleic acids.
  • the term "enzymatic manipulation of nucleic acids” used here refers to any enzymatically catalyzed reaction in which nucleic acids, preferably DNA or RNA, are enzymatically changed. These include polymerization reactions, for example the polymerase chain reaction (PCR) or the ligase chain reaction (LCR), ligation reaction, phosphorylation reactions , Dephosphorylation reactions, coupling reactions, preferably labeling reactions of nucleic acids with detectable markers, for example the addition of radioactive or non-radioactive labels at the 5 ' or 3 ' end of an RNA or DNA, the fragmentation of DNA, for example via restriction reactions, the preferred enzymes Polymerases, ligases, kinases, nucleases, restriction endonucleases and transferases are preferably the mixture or solution according to the invention is used when carrying out a polymerase chain reaction moderate mixture or the solution for neutralizing inhibitors of thermostable DNA polymerases, for example Taq DNA polymerase, is used.
  • the present invention further relates to reaction vessels which are characterized in that they have a coating with the mixture according to the invention or have been coated with the solution according to the invention.
  • the reaction vessels according to the invention can consist of different materials, preferably those materials as are used for the production of reaction vessels that are used for the enzymatic manipulation of nucleic acids.
  • the reaction vessels preferably consist of polypropylene, polyethylene, polycarbonate, polystyrene or borosilicate.
  • reaction vessels according to the invention are preferably 0.2 ml-0.5 ml or 0.65 ml reaction vessels in the form known to the person skilled in the art (for example in the form of so-called “eppendorft tubes”), strips with 8 or 12 single-use reaction vessels connected to one another a '200 ⁇ l, for plates with 12, 24, 48, 96, 192, 384, 768 or 1536 cavities, for glass capillaries or for silicon “wafers” with up to several thousand reaction compartments.
  • the reaction vessels according to the invention are additionally containing enzymes such as. B. coated polymerases, ligases or nucleases, which are involved in the desired manipulation of the nucleic acid.
  • enzymes such as. B. coated polymerases, ligases or nucleases, which are involved in the desired manipulation of the nucleic acid.
  • the reaction vessels can, for example, already be coated with NTPs.
  • This is preferably a thermostable “hot start” Taq polymerase, the polymerase activity of which is reversibly inhibited by the occupation of the nucleotide binding region by antibodies or aptamers until the first heat denaturation.
  • reaction vessels with the coating described above can be carried out by a person skilled in the art using standard methods, depending on the material of the reaction vessel walls. So can for example, proceed as follows:
  • the antibody NF5E6 was deposited with the DMSZ on January 12, 2000 (accession number DSM ACC2436). With the same results, the antibody EDD1D9 can be used analogously to NF5E6. This antibody was also deposited on January 12, 2000 at the DSMZ with the accession number DSM ACC2435. example 1
  • PCR reaction tubes were used for coating: Advanced Biotechnologies LTD, Cat. #. AB-0350, 0.5 ml PCR reaction tubes.
  • Antibody NF5E6 of connex, 5 ⁇ g / 25 ⁇ l reaction volume spermidine: Sigma, order no. S-0266, 5 ⁇ g / 25 ⁇ l reaction volume BSA: Boehringer Mannheim, order no. 711,454, 10 ⁇ g / 25 ⁇ l
  • Reaction volume This mixture was applied to the bottom of the reaction vessels in the area of the usable space depending on the intended 25 ⁇ l reaction volumes.
  • the tubes were dried for 40 h in a "Weiss" climatic chamber at 38 ° C +/- 1 ° C. After 24 h there was a relative humidity of ⁇ 2%. Finally, the reaction vessels were closed and packed in a moisture-proof manner.
  • PCR reaction tubes were used: Advanced Biotechnologies LTD, Cat. #. AB-0350, 0.5 ml PCR reaction tubes
  • reaction tubes were coated as follows: Antibody: NF5E6 from Connex, 5 ⁇ g / 25 ⁇ l reaction volumespermidine: Sigma, order no. S-0266, 5 ⁇ g / 25 ⁇ l reaction volume
  • BSA Boehringer Mannheim, order no. 711454, 10 ⁇ g / 25 ⁇ l
  • Hp-R1284 (5 'CCT GTT AGC AAC TAA GAA AGG 3')
  • Each DNA sample was placed in reaction vessels with and without a coating
  • the amplified fragments were detected by means of electophoretic separation in an agarose gel (2%). This electrophoretic separation produces characteristic bands of the same fragment length. These bands were stained with ethidium bromide (Merck) and made visible in ultraviolet light .
  • the inhibitor was used as follows:
  • Hemin Sigma, 1 ⁇ g in 25 ml reaction volume
  • the neutralizers were used as follows:
  • Antibody NF5E6 from Connex, 20 ⁇ g in 25 ⁇ l reaction volume (addition in liquid form)
  • BSA Boehringer Mannheim, order number 711454, 20 ⁇ g in 25 ⁇ l
  • Target DNA used Helicobacter pylori DNA
  • Hp-R1284 (5 ' CCT GTT AGC AAC TAA GAA AGG 3 ' )
  • the neutralizer spermidine shows no positive effect.
  • the neutralizers antibody and BSA show a positive effect.
  • the positive effect of the individual components is significantly exceeded by using the coating with the mixture according to the invention.
  • the inhibitor was used as follows:
  • the neutralizers were used as follows:
  • Antibody NF5E6 from Connex, 20 ⁇ g in 25 ⁇ l reaction volume (addition in liquid form)
  • BSA Boehringer Mannheim, order number 711454, 20 ⁇ g in 25 ⁇ l
  • Thermocycler used Biozym MJ, Research PTC 200 Target DNA used: Helicobacter pylori DNA Gene area: 16s rRNA gene
  • the use of BSA or antibodies alone shows no abolition of the inhibitory effect of heparin.
  • the inhibitory effect of heparin on the amplification can only be eliminated by adding spermidine or using the mixture according to the invention.
  • the use of the mixture according to the invention leads to a significantly stronger neutralization of the inhibitory effect of the heparin than the use of the single component spermidine.
  • Gall salts Sigma, order number: B8756, 10 ⁇ g in 25 ⁇ l reaction volume
  • the neutralizers were used as follows:
  • Antibody NF5E6 from Connex, 20 ⁇ g in 25 ⁇ l reaction volume (addition in liquid form)
  • BSA Boehringer Mannheim, order number 711454, 20 ⁇ g in 25 ⁇ l
  • Thermocycler used Biozym MJ, Research PTC 200
  • Target DNA used Helicobacter pylori DNA
  • BSA and spermidine show no neutralization of the inhibitory effect of gall salts.
  • the addition of antibody or the addition of the mixture according to the invention have a neutralizing effect on the inhibitory effect of the gall salts.
  • the neutralizing effect of the mixture according to the invention but is significantly stronger than the effect of the single component antibody.
  • PCR reaction tubes were used: Advanced Biotechnologies LTD, Cat. #. AB-0350, 0.5 ml PCR reaction tubes
  • the coating of the reaction vessels was as follows: Antibody: NF5E6 of connex, 05 ⁇ g / 25 ⁇ l reaction volume spermidine: Sigma, order no. S-0266, 05 ⁇ g / 25 ⁇ l reaction volume BSA: Boehringer Mannheim, order no. 711,454, 10 ⁇ g / 25 ⁇ l reaction volume
  • Thermo Cycler used Biometra - Personal Cycler, Vers. 3.01 bc, (c) Biotron
  • Hp-R1284 (5 'CCT GTT AGC AAC TAA GAA AGG 3') PCR components used:
  • the coating enables the target microorganism to be detected at lower DNA concentrations (two orders of magnitude earlier than without a coating).
  • the NF5E6 antibody in the mixture according to the invention is interchangeable with other IgG antibodies.
  • different IgG antibodies were mixed with BSA and spermidine in the same concentrations as in the 10 ⁇ mixture according to the invention and used in the PCR reaction.
  • a direct sex determination on human heparinized whole blood without previous DNA extraction was chosen as the test system, since whole blood contains a high concentration of inhibitors.
  • Molecular biological gender determination is based on the detection of a gender-specific gene, which is located on the X or Y chromosome. If only the female gene can be detected, it is a female individual (two X chromosomes), but if both genes are found, it is a male individual (one X and one Y in the male).
  • Antibodies NF5E6, Connex GmbH, 2mg / mL
  • PCR reaction vessels were used: Abgene LTD, Cat. # AB-0773, 0.2mL flat caps; Thermo Cycler used: Biometra - Personal Cycler, Vers. 3.01 bc, serial no .: 9510443; Sample material used for DNA amplification: human heparinized whole blood; Blood collection system used: Sarstedt Monovette, Li-Heparin 10-30IE / mL, order no. 06 / 1565.001, Sarstedt cannula, GX1, order no .: 85 / 1,160;
  • Amplified gene regions amplification of the X and Y-specific single copy gene amelogenin (AMG); Gene, 167 (1995) 327-332. Length of the amplified fragments: X chromosome 330 bp
  • AMG-Universal (AMG-univ., 5 ' -CAGCTTCCCAGTTTAAGCTCT-3 ' )
  • AMG-X (AMG-X, 5 ' -TCTCCTATACCACTTAGTCACT-3 ' )
  • AMG-Y (AMG-Y, 5 , -GCCCAAAGTTAGTAATTTTACCT-3 , )
  • the antibody NF5E6 in the mixture according to the invention was replaced by another IgG antibody.
  • the other IgG antibodies were used in the PCR reactions with BSA and spermidine in the same concentration as the antibody NF5E6.
  • the multiplex polymerase chain reaction is a PCR variant in which two or more loci are amplified simultaneously in one PCR reaction.
  • the current method is based on the amplification of the single copy amelogenin gene (AMG).
  • AMG single copy amelogenin gene
  • the gene of the Y allele has a deletion in the first intron compared to that of the X allele. This deletion enables specific PCR reactions to differentiate between X and Y alleles. Amplification with three primers, two of which are allele-specific, allows unique identification of both the X and Y alleles in a single PCR reaction.
  • the 5'-primer (HAMG-univ, 5'-CAGCTTCCCAGTTTAAGCTCT-3 ') an amplification of the X and the Y-AMG allele is possible.
  • the 3 'primers are specific for both the X and the Y allele.
  • the 3 'X-specific primer (HAMG-X, 5'-TCTCCTATACCACTTAGTCACT-3') is derived from the sequence deleted in the Y chromosome and the 3'Y-specific primer (HAMG-Y, 5'- GCCCAAAGTTAGTAATTTTACCT-3 ' ) covers the area that connects the two deletion endpoints.
  • the 330 bp (X chromosome) and the 218 bp (Y chromosome) PCR products are then analyzed using agarose gel electrophoresis. Usually three primers are used simultaneously in one reaction in a reaction vessel.
  • Heparinized whole blood (heparin concentration 10-30 IU / ml) was used for the PCR reaction.
  • the nucleated cells present in the blood served as a DNA resource; the heparinized blood was also a complex mixture of inhibitors (eg: hemoglobin, heme, heparin, etc.).
  • the blood was diluted with H 2 0 (0, 1: 5, 1: 10, 1: 20) and 1 ⁇ L of each was added to 19 ⁇ L master mix.
  • An approach without inhibitors served as a positive control; 10ng of human genomic DNA (male) were used as the template.

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  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physics & Mathematics (AREA)
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  • Sustainable Development (AREA)
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  • Medicinal Chemistry (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Abstract

L'invention concerne un mélange constitué d'albumine et/ou de spermidine et d'au moins un anticorps, de préférence un mélange d'albumine, de préférence d'albumine sérique bovine, de spermidine et d'un anticorps, ou une solution contenant ledit mélange. Ce mélange peut être utilisé pour produire un tampon réactionnel ou pour revêtir des réacteurs, afin de neutraliser les substances qui inhibent la manipulation d'enzymes utilisées pour la manipulation d'acides nucléiques et sont présentes normalement dans les échantillons. Ce mélange permet d'accroître fortement l'efficacité de modifications d'acides nucléiques catalysées par voie enzymatique, par exemple la réaction en chaîne de la polymérase (PCR), ou permet même de réaliser ce type de réactions dans le cas de certains échantillons. Enfin, l'invention concerne également un procédé de production desdits réacteurs.
PCT/EP2000/003933 1999-04-30 2000-05-02 Melange pour neutraliser des inhibiteurs d'enzymes Ceased WO2000071698A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU47543/00A AU4754300A (en) 1999-04-30 2000-05-02 Mixture for neutralizing enzyme inhibitors

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19919942 1999-04-30
DE19919942.6 1999-04-30

Publications (2)

Publication Number Publication Date
WO2000071698A2 true WO2000071698A2 (fr) 2000-11-30
WO2000071698A3 WO2000071698A3 (fr) 2001-02-15

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PCT/EP2000/003933 Ceased WO2000071698A2 (fr) 1999-04-30 2000-05-02 Melange pour neutraliser des inhibiteurs d'enzymes

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Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3416981B2 (ja) * 1993-03-30 2003-06-16 株式会社島津製作所 核酸合成法
CA2202990A1 (fr) * 1994-10-21 1996-05-02 Alan H. Davis Emploi de spermidine pour attenuer les effets inhibiteurs de reactions en chaine de ligase dans un echantillon d'essai clinique
AU4775497A (en) * 1996-09-19 1998-04-14 W. Kurt Roth Method for purifying and eventually analyzing nucleic acids from biological test samples

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AU4754300A (en) 2000-12-12
WO2000071698A3 (fr) 2001-02-15

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