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WO2003066890A2 - Procede permettant de determiner le modele de methylation d'adn - Google Patents

Procede permettant de determiner le modele de methylation d'adn Download PDF

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Publication number
WO2003066890A2
WO2003066890A2 PCT/EP2003/001035 EP0301035W WO03066890A2 WO 2003066890 A2 WO2003066890 A2 WO 2003066890A2 EP 0301035 W EP0301035 W EP 0301035W WO 03066890 A2 WO03066890 A2 WO 03066890A2
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WO
WIPO (PCT)
Prior art keywords
dna
detector
sequences
hybridization
dna sequences
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PCT/EP2003/001035
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German (de)
English (en)
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WO2003066890A3 (fr
Inventor
Jochen Muth
Andreas Kappel
Heike Behrensdorf
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Nanogen Recognomics Gmbh
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Filing date
Publication date
Application filed by Nanogen Recognomics Gmbh filed Critical Nanogen Recognomics Gmbh
Priority to AU2003210200A priority Critical patent/AU2003210200A1/en
Publication of WO2003066890A2 publication Critical patent/WO2003066890A2/fr
Publication of WO2003066890A3 publication Critical patent/WO2003066890A3/fr

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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

Definitions

  • the present invention relates to a method for determining the methylation pattern of DNA, in particular using electronically addressable biochips.
  • the methylation of DNA plays an important role in nature.
  • the activity of genes is regulated with the help of the methylation of individual cytosines in the DNA.
  • the methylation rate and the methylation pattern in DNA there is great interest in methods with which such methylations can be easily detected. For this reason, several methods have now been developed which are suitable for determining the methylation rate and the methylation pattern in DNA.
  • restriction enzymes that cannot recognize methylated sites as substrates (e.g. described in Analysis of DNA methylation and DNAse I sensitivity in gene-specific regions of chromatin; Ginder, G ⁇ rdon; Methods Hematol. Vol. 20 1989 p. 111 -123). With these methods, however, only methylations that occur at the restriction sites can be detected.
  • methylation of DNA sequences is determined using PCR (MethylLight: a high-througput assay to measure DNA methylation; Cindy A. Eads, Kathleen D. Danenberg, Kazuyuki Kawakami, et. Al .; Nucleic Acids Rearch 2000 Vol. 28. No 8.).
  • PCR Method of PCR
  • a corresponding dye-labeled sample must be synthesized for each methylation position, which is technically very complex.
  • the methylation of DNA can also be determined quantitatively by chemical reactions with chloroacetaldehyde.
  • the detection of methylation is carried out in this method using a spectrometer in the range between 300 and 400 nm.
  • the methylation pattern is determined using bisulfite SSCP, whereby unmethylated cytosine is converted to uracil.
  • the sequence of the DNA sample examined can then be determined with the aid of a DNA sequencer (Quantitative DNA Methylation analysis by fluorescent ploymerase chain reaction Single Strand conformation polymorphism using an automated DNA sequencer; Hiromu Suzuki, Fumio Itoh, Minoru Toyota Tekefumi KiKuchi Electrophoresis 200021, 904-908).
  • the invention is therefore based on the object of providing a simple method for determining the methylation pattern of DNA sequences.
  • the task is solved by a process that comprises the following process steps:
  • starting DNA means a DNA or a DNA fragment with a known sequence, the methylation pattern of which is to be determined.
  • ctor DNA includes DNA polynucleotides that are complementary to partial sequences of the starting DNA sequence, all guanine bases preferably being replaced by adenine bases.
  • spatially resolved fixation means that a specific detector DNA sequence can be assigned a defined location on a carrier.
  • the carrier can be loaded passively, for example using pipetting devices or actively, via electronic addressing Methods for the production of such carriers are known to the person skilled in the art, and corresponding methods for producing electronically addressable carrier surfaces are described, for example, in Radtkey et al., Nucl. Acids Res. 28, 2000, e17; RG Sonowsky et al., Proc. Natl. Acad. Sci. USA, 1119-1123; 1994 PN Gilles et al., Nature Biotechnol. 17, 365-370, 1999, the production itself also being able to be carried out by means of electronic addressing.
  • the starting DNA to be examined can e.g. B. a gene or gene fragment isolated from the cell nucleus, the nucleotide sequence of which is known.
  • the starting DNA can be further fragmented for better handling.
  • the fragmentation can be unspecific e.g. B. by shear forces or specifically z. B. done by a restriction digest.
  • the DNA fragments thus obtained can then be denatured for chemical conversion.
  • the chemical conversion of unmethylated cytosine in the starting DNA sequences is preferably carried out using bisulfites, whereby Methylcytosine is not implemented in the reaction.
  • the DNA sequence treated in this way can now be hybridized with a detector DNA sequence, it being possible for the detector DNA sequence to be complementary to the chemically untreated starting sequence.
  • the hybridization of the chemically treated DNA sequence with the detector DNA sequence consequently results in guanine / uracil mismatches at the sequence positions at which an unmethylated cytosine was present in the starting sequence.
  • detector DNA sequences are used whose sequence is complementary to the chemically untreated starting sequence, the guanine bases being replaced by adenine bases. Adenine / methylcytosine mismatches are thus obtained when the detector DNA sequence is hybridized with the chemically treated DNA sequence.
  • This variant of the method is particularly suitable for determining the methylation pattern of low-methylated DNA sequences, as are usually found in mammals or plants. Mammalian DNA has a methylation rate of 3 to 10%, that of plants up to 50%. Viruses, on the other hand, have a degree of methylation of up to 100%.
  • the direct detection of the non-methylated cytosines via the corresponding guanine / uracil mismatches can also be advantageous. It is also possible to carry out both process variants in parallel.
  • detector DNA sequences z. B synthetically accessible polynucleotides, preferably with a length between 5 and 100, particularly preferably between 12 and 35 nucleotides, can be used.
  • the detector DNA sequences can be derived directly from the known starting DNA sequence.
  • the individual detector DNA sequences can overlap, so that ultimately a mismatch that occurs can always be assigned to a specific location within the starting DNA sequence.
  • the mismatches can finally be identified with mismatching substances, such as. B. mutS can be demonstrated.
  • the chemical conversion of non-methylated cytosine bases in DNA sequences is preferably carried out on single strands which, for. B. can be obtained by denaturing double-stranded DNA samples in alkaline. The converted DNA can then, if necessary, be worked up further, ie separated, denatured, neutralized, precipitated and desalted.
  • the DNA to be converted containing methylated cytosine bases is dissolved in water and then in the alkaline solution by adding a sodium bisulfite hydroquinone solution elevated temperature with exclusion of light after the reaction is complete, the DNA obtained can be purified by chromatography.
  • the hybridization of the chemically converted DNA nucleotides with the detector DNA can take place on any surface to which the hybrids can be bound in a spatially resolved manner.
  • the hybridization itself can take place passively or actively electronically accelerated, as z. B. is possible with a chip from Nanogen Inc. (San Diego / USA).
  • the electronic addressing is carried out by applying an electrical field, preferably between 1.5 V and 2.5 V with an addressing time between 1 and 3 minutes. Due to the electrical charge of the nucleotide sequences to be addressed, their migration is greatly accelerated by applying an electric field.
  • the addressing can take place in a spatially resolved manner, addressing being carried out successively at different zones of the chip surface. You can send to the different addressing and hybridization conditions can be set in individual locations.
  • both the guanine / uracil and the adenine / methylcytosine mismatches can be detected with substrates that recognize mismatches.
  • suitable substrates are e.g. B. base mismatch binding proteins such as mutS or mutY, preferably from E.coli, T. thermophilus or T.aquaticus, MSH 1 to 6, preferably from S.cerevisiae, S1 nuclease, T4 endonuclease, thymine cosylase, cleavase or fusion proteins containing one Domain of these base mismatch binding proteins.
  • the protein which recognizes mismatches can also contain polymeric markers (J. Biotechnol. 35, 165-189, 1994), metal markers, enzymatic or radioactive markings or quantum dots (Science Vol 281, 2016, Sep 25, 1998 ) contain.
  • the enzyme label can z. B. be a direct enzyme coupling or an enzyme substrate transfer or an enzyme complementation. Chloramphenicol acetyl transferase, alkaline phosphatase, luciferase or peroxidase are particularly suitable for enzymatic labeling.
  • substrate marking with dyes which absorb or emit light in the range between 400 and 800 nm is preferred.
  • fluorescent dyes suitable for labeling are especially Cy TM 3, Cy TM 5 (from Amersham Pharmacia), Oregon Green 488, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 594, Alexa Fluor 647, Bodipy 558/568 , Bodipy 650/665, Bodipy 564/570 (e.g. from Mobitec, Germany), S 0535, S 0536 (e.g. from FEW, Germany), Dy-630-NHS, Dy-635 -NHS, EVOblue30-NHS (e.g.
  • labeled antibodies can also be used directly against mutS or against a fused peptide domain, e.g. B. MBP are directed.
  • the fluorescence signals obtained can be directly translated into the corresponding methylation pattern in the cases in which exactly one possible mismatch can occur at each location on the support.
  • several potential mismatch positions cytosines in the corresponding partial sequence of the starting DNA
  • detector DNA sequences which overlap in their sequence. The evaluation is then carried out according to the “clustering analysis” method (described in Eisen, MB, Spellman, PT, Brown PO, Botstein D 1998, Cluster analysis and display of genome wide expression pattern, Proc. Natl.
  • the specificity of the measurement of mismatches with mutS, a preferred mismatching substrate, can be further increased by the addition of surfactants or by the addition of BSA that blocks non-specific binding sites on the hybrids.
  • MutS also has the advantage that measurements can also be carried out under low salt conditions up to a salt concentration of 10 mM.
  • any single-stranded nucleotide sequences fixed on the support after the hybridization step can be broken down by adding a nuclease, preferably a mung bean nuclease or S1 nuclease.
  • a nuclease preferably a mung bean nuclease or S1 nuclease.
  • the methylation pattern of DNA fragments with known sequences can be determined simply and quickly.
  • the measuring time can be reduced further and the reliability of the measurement can be improved by the very good electrical controllability of the hybridization.
  • the carriers once covered with detector DNA are also reusable, the measured DNA can simply be dehybridized and washed off the surface. This enables a high sample throughput with an occupied carrier.
  • Step I) shows the chemical conversion of starting DNA oligonucleotides.
  • the oligonucleotides are shown in dashed lines, the left oligonucleotide containing a methylcytosine ( 5m C), the right oligo in the same place an unmethylated cytosine (C).
  • the unmethylated cytosine bases are converted into uracil.
  • the oligonucleotides are then applied to a chip surface which contains detector DNA with an adenine base at the position on the detector DNA oligonucleotide which is complementary to the 5m C and C positions (step II)).
  • Step III) shows the hybridization of the applied oligonucleotides with the detector DNA sequence, wherein in the presence of a methylcytosine at this position a mismatch occurs which can be detected with fluorescent-labeled mutS.
  • the oligonucleotides Seq. ID No. 1, 2 and 3 (0.05 ⁇ mol scale) diluted with water so that a final concentration of 10 picomoles / 1 ⁇ l is obtained.
  • the aqueous solution was then dissolved in 50 mM histidine buffer 1: 100.
  • the methyl modified bases are marked as 5m C.
  • the reaction is completed by adding 3 M NaOH to the oligonucleotide fraction obtained to a final NaOH concentration of 0.3 M at 37 ° C.
  • the chemically converted DNA oligonucleotides obtained are then purified again by chromatography on a NAP5 column and desalted. 50 ul of the desalted solution were diluted with 50 ul 100 mM histidine solution. DNA oligonucleotides are obtained whose unmethylated cytosine bases have been converted into uracil.
  • the corresponding Alexa Fluor 647 succinimidyl ester is non-specifically covalently linked to protein lysine residues via a nucleophilic substitution reaction in accordance with the FluoroLink TM product specification protocol, Amersham Pharmacia Biotech, Little Chalfont, UK.
  • the fluorescence labeling of mutS is carried out by incubation with a large excess of fluorophore under strongly basic conditions (0.1 M Na 2 C03, pH 9.3).
  • the fluorescence-labeled mutS obtained is purified by gel permeation chromatography.

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Abstract

Procédé permettant de déterminer le modèle de méthylation dans des séquences d'ADN, selon lequel la cytosine non méthylée de la séquence d'ADN est convertie chimiquement en uracile et la séquence d'ADN ainsi traitée est hybridée avec une séquence d'ADN détectrice, les hybrides étant fixés avec résolution locale sur une surface. Finalement, les mésappariements guanile / uracile sont détectés à l'aide de substances reconnaissant lesdits mésappariements.
PCT/EP2003/001035 2002-02-04 2003-02-03 Procede permettant de determiner le modele de methylation d'adn WO2003066890A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003210200A AU2003210200A1 (en) 2002-02-04 2003-02-03 Method for determining the methylation pattern of dna

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE2002104566 DE10204566A1 (de) 2002-02-04 2002-02-04 Verfahren zur Bestimmung des Methylierungsmusters von DNA
DE10204566.6 2002-02-04

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WO2003066890A3 WO2003066890A3 (fr) 2004-03-25

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102004002257B4 (de) * 2004-01-09 2006-11-09 Epigenomics Ag Verfahren zur Untersuchung von Cytosin-Methylierungen in DNA mit Hilfe von DNA-Reparaturenzymen

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999028498A2 (fr) * 1997-11-27 1999-06-10 Epigenomics Gmbh Procede de production d'empreintes de doigt complexes a methylation d'adn
WO1999039003A1 (fr) * 1998-01-30 1999-08-05 Genzyme Corporation Procede de detection et d'identification de mutations
US6051380A (en) * 1993-11-01 2000-04-18 Nanogen, Inc. Methods and procedures for molecular biological analysis and diagnostics
EP1041160A1 (fr) * 1997-07-31 2000-10-04 The Institute Of Physical & Chemical Research Procede de detection de mutation dans une sequence de bases
WO2001044504A2 (fr) * 1999-12-17 2001-06-21 Astrazeneca Ab Methode diagnostique

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5849486A (en) * 1993-11-01 1998-12-15 Nanogen, Inc. Methods for hybridization analysis utilizing electrically controlled hybridization
DE19853398C1 (de) * 1998-11-19 2000-03-16 Epigenomics Gmbh Verfahren zur Identifikation von Cytosin-Methylierungsmustern in genomischer DNA

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6051380A (en) * 1993-11-01 2000-04-18 Nanogen, Inc. Methods and procedures for molecular biological analysis and diagnostics
EP1041160A1 (fr) * 1997-07-31 2000-10-04 The Institute Of Physical & Chemical Research Procede de detection de mutation dans une sequence de bases
WO1999028498A2 (fr) * 1997-11-27 1999-06-10 Epigenomics Gmbh Procede de production d'empreintes de doigt complexes a methylation d'adn
WO1999039003A1 (fr) * 1998-01-30 1999-08-05 Genzyme Corporation Procede de detection et d'identification de mutations
WO2001044504A2 (fr) * 1999-12-17 2001-06-21 Astrazeneca Ab Methode diagnostique

Non-Patent Citations (3)

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Title
GOTOH M ET AL: "Rapid method for detection of point mutations using mismatch binding protein (MutS) and an optical biosensor" GENETIC ANALYSIS: BIOMOLECULAR ENGINEERING, ELSEVIER SCIENCE PUBLISHING, US, Bd. 14, Nr. 2, 1. Juli 1997 (1997-07-01), Seiten 47-50, XP004126266 ISSN: 1050-3862 *
REIN ET AL: "Identifying 5-methylcytosine and related modifications in DNA genomes" NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, SURREY, GB, Bd. 26, Nr. 10, 1998, Seiten 2255-2264, XP002143106 ISSN: 0305-1048 *
WAGNER R ET AL: "MUTATION DETECTION USING IMMOBILIZED MISMATCH BINDING PROTEIN (MUTS)" NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, SURREY, GB, Bd. 23, Nr. 19, 1995, Seiten 3944-3948, XP002916167 ISSN: 0305-1048 *

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WO2003066890A3 (fr) 2004-03-25
DE10204566A1 (de) 2003-08-14
AU2003210200A1 (en) 2003-09-02

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