EP1366190A2 - Procede pour quantifier des methylations par cytosine dans de l'adn genomique amplifie de maniere complexe - Google Patents

Procede pour quantifier des methylations par cytosine dans de l'adn genomique amplifie de maniere complexe

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Publication number
EP1366190A2
EP1366190A2 EP01989384A EP01989384A EP1366190A2 EP 1366190 A2 EP1366190 A2 EP 1366190A2 EP 01989384 A EP01989384 A EP 01989384A EP 01989384 A EP01989384 A EP 01989384A EP 1366190 A2 EP1366190 A2 EP 1366190A2
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EP
European Patent Office
Prior art keywords
dna
sample
adapters
amplified
genomic dna
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.)
Withdrawn
Application number
EP01989384A
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German (de)
English (en)
Inventor
Alexander Olek
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Epigenomics AG
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Epigenomics AG
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Filing date
Publication date
Application filed by Epigenomics AG filed Critical Epigenomics AG
Publication of EP1366190A2 publication Critical patent/EP1366190A2/fr
Withdrawn legal-status Critical Current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
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
    • 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
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation

Definitions

  • the invention relates to a method for the quantification of cytosine methylations of a genomic DNA sample with an unknown methylation status by comparison with a demethylated reference DNA.
  • 5-Methylcytosine is the most common covalently modified base in the DNA of eukaryotic cells. For example, it plays a role in the regulation of transcription, genomic imprinting and in tumorigenesis. The identification of 5-methylcytosine as a component of genetic information is therefore of considerable interest. However, 5-methylcytosine positions cannot be identified by sequencing since 5-methylcytosine has the same base pairing behavior as cytosine. In addition, in the case of PCR amplification, the epigenetic information which the 5-methylcytosines carry is completely lost.
  • a chemical reaction or enzymatic treatment of the genomic DNA is usually carried out, as a result of which the cytosine can be distinguished from the methylcytosine bases.
  • a common method is the implementation of genomic DNA with bisulfite, which is alkaline
  • Demethylated DNA is used in the prior art in numerous methods for quantifying DNA methylation. Representative are: “Rapid quantification of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension” (Gonzalgo, ML, Jones PA Nucleic Acids Res. 25, 2529 (1997)) and “Detection and measurement of PCR bias in quantitative methylation analysis of bisulphite-treated DNA “(Warnecke, PM, Stirzaker, C, Melki, JR, Douglas, SM, Paul, CL, Clark, SJ 25, 4422 (1997)).
  • This demethylated DNA is e.g. obtained from cells that are demethylated in the target sequence or from cells that lack the enzyme DNA methyltransferase.
  • this method is not suitable for carrying out complex amplifications which are intended to provide many fragments at the same time for the methylation detection.
  • this is essential since the sample to be examined, for the methylation analysis of which the reference DNA is produced, is usually only amplified after the bisulfite treatment. In the case of a complex PCR reaction, the comparability of reference and sample is no longer possible, since they contain potentially different fragments.
  • the present invention provides a method for the analysis of cytosine methylations in genomic DNA samples, for which purpose DNA with a degree of methylation of 0% is produced as reference material. This is the first time possible to provide an essentially unmethylated reference DNA for complex amplifications.
  • the present invention describes a method for providing demethylated DNA as reference material for the analysis of cytosine methylations in genomic DNA samples using complex amplifications.
  • the following process steps are carried out in detail:
  • a genomic DNA sample is amplified with primers which are either very short or degenerate oligonucleotides or oligonucleotides complementary to adapters.
  • primers which are either very short or degenerate oligonucleotides or oligonucleotides complementary to adapters.
  • a restriction enzyme is cut before amplification and the adapters, which are short nucleotide fragments of known sequence, are ligated to the ends of the resulting DNA fragments.
  • the amplificates are treated chemically in such a way that at the 5-position unmethylated cytosine bases are converted into uracil, thymine or into another base which is unlike the cytosine in hybridization behavior, while the 5-methylcytosine bases remain essentially unchanged. This is understood below as chemical pretreatment.
  • the chemically pretreated amplificates are in turn amplified. Either several specifically hybridizing oligonucleotides or oligonucleotides complementary to the adapters are used as primers. In the latter case, chemical pretreatment is also carried out.
  • a genomic DNA sample to be examined is cut using a restriction enzyme. Adapters are ligated to the ends of the DNA fragments and the sample is subsequently divided. The first part of the sample is amplified with primer oligonucleotides that are complementary to the adapters. However, the second part of the sample is not amplified.
  • the two parts of the sample are chemically pretreated and amplified separately, using primer oligonucleotides complementary to the adapters.
  • the two parts of the sample are then analyzed.
  • the first part of the sample provides the reference value for a degree of methylation of 0%.
  • the second part of the sample provides the measured value which essentially corresponds to the degree of methylation in the original genomic DNA sample.
  • the genomic DNA to be analyzed is preferably obtained from the usual sources for DNA, such as. B. cell lines, blood, sputum, stool, urine, brain spinal fluid, paraffin-embedded tissue, for example tissue from the eyes, intestine, kidney, brain, heart, prostate, lungs, chest, liver, skin or bone marrow, histological slides and all possible combinations of them.
  • the polymerase chain reaction (PCR) is used for the amplification.
  • PCR polymerase chain reaction
  • a heat-resistant DNA polymerase is used for the polymerase chain reaction.
  • the amplification of several identical or several different DNA sections is preferably carried out in a reaction vessel.
  • restriction endonucleases The following are preferably used as restriction endonucleases: Rsal, Dpnl, Dpnll, Msel, Sau3AI, Alul, NIalll, Hael- II, Bfal, Tsp509I, BstUI or Mbol.
  • the amplificates are separated from the reagents and other constituents of the reaction mixture by binding to a solid phase or to a gel and washing steps.
  • the reagents and the other constituents of the reaction mixture are preferably diluted in such a way that they are no longer a hindrance in the subsequent amplification, but the concentration of the amplified treated is still sufficient for the second amplification.
  • the demethylated reference DNA produced is particularly preferably analyzed in the same way as a sample DNA to be examined. In the analysis, this reference DNA provides the comparison value for a degree of methylation of 0%.
  • an enzymatically methylated DNA which was treated in the following in the same way as the sample DNA, is preferably used as a reference for a degree of methylation of 100%.
  • Example la Preparation of a demethylated reference DNA by means of multiplex PCR
  • the following example relates to the preparation of a downmethylated DNA sample that serves as a reference in comparison to an unknown methylated DNA.
  • you uses a genomic DNA sample, which in this case was digested with the restriction enzyme, Mssl.
  • (1-40 ng) of the cut DNA are multiplied by a pre-amplification by a DOP-PCR (degenerate oligonucleotide primed polymerase chain reaction) according to the Nelson method (VG Cheung, SF Nelson, PNAS 93, 1476-1479, 1996) with the genomic primer oligonucleotide 5 '-CCGACTCGAGNNNNNNATGTG G-3'.
  • the method is primarily used to pre-amplify very small amounts of genomic DNA in order to allow multiple genetic analysis from 2-15 ⁇ g (200-1000 bp). In the amplification, all methyl cytosines are treated as cytosine bases.
  • the PCR reaction is carried out in the Master Cycler Gradient (Eppendorf, Hamburg) with the following program.
  • the bisulfite reaction leads to the conversion of all cytosine bases into uracil.
  • To purify the bisulfited DNA it is bound to a reversed phase C18 solid phase and freed of chemicals by washing with a suitable buffer solution. Then the DNA with a polar solvent such as. B.
  • the specific amplification is carried out with 128 primer oligonucleotides, at least 64 primer oligonucleotides being labeled with Cy5 (Amersham Pharmacia). This is a primer oligonucleotide from a pair of primers.
  • primer oligonucleotide mixture (128 primer oligonucleotides, 64 of which are labeled Cy5, 0.78 pmol / ul of each)
  • the PCR reaction is carried out in the Master Cycler Gradient (Eppendorf, Hamburg) with the following program. 15 min 95 ° C
  • the generated PCR certificates were analyzed by agarose gel electrophoresis (1.5% agarose in 0.5 ⁇ TBE buffer, Sambrook et al.). For this, 4 ⁇ l of the PCR approach are subjected to gel electrophoresis. Under the specified conditions, 64 genes are successfully amplified simultaneously.
  • Example Ib Preparation of an unknown methylated DNA sample by means of multiplex PCR
  • example Ib relates to the preparation of an unknown methylated DNA sample, which is compared with the downmethylated reference DNA from example la.
  • a genomic DNA sample is used, which in this case was cleaved with the restriction enzyme Mssl.
  • the sample is then reacted with hydrogen sulfite (bisulfite, disulfite).
  • the first method (Olek et al., Nucl. Acids. Res. 1996, 24, 5064-5066) is a reaction with hydrogen sulfite and a radical scavenger, the DNA being embedded in agarose. The desulfonation of the DNA also takes place in agarose.
  • An organic reagent that supports denaturation is added and the mixture is incubated at elevated temperature.
  • hydrogen sulfite all cytosine bases are converted to uracil in both methods, methyl cytosines being retained.
  • a polar solvent such as. B.
  • the preamplification of the hydrogen sulfite-treated DNA is carried out using degenerate primer oligonucleotides (5 '-TTATAATGTTTT and 5' -TATATACTAAT).
  • the subsequent amplification with Cy5-labeled bisulfite-specific primer oligonucleotides is carried out with the 128 primer oligonucleotides described in Example 1a, the same primer oligonucleotide being Cy5-labeled.
  • the amplificates are also subjected to agarose gel electrophoresis for analysis.
  • Example lc Comparison of the unknown methylated DNA sample with the downmethylated reference DNA
  • the comparison of the unknown methylated DNA sample with the downmethylated reference DNA is preferably carried out by hybridization to an oligonucleotide array. Fluorescent dots are visible according to the position on the array. It is noticeable that certain points on the array are relative to the others and to the reference DNA shows a significantly increased or decreased fluorescence, provided the amplified products are present in a comparable concentration in the individual samples to be examined. The intensities of the fluorescent dye Cy5 (635 nm) in the individual amplificates are measured. The manner in which fluorescence measurements are evaluated is known to the person skilled in the art.
  • a genomic sequence is enzymatically cleaved by adding a restriction enzyme, here Nallall (Fermentas), which recognizes the sequence CATG. Fragments with an average size of 400 bp are generated. The cleaved fragments have 3 'overhanging CATG ends and are ligated to the sequence sections with the oligomer with the genomic sequence TGTCATCCTGTTGTCATG with addition of T4-DNA ligase according to standard conditions (fermentas) and non-ligated adapters according to standard conditions with a cleaning kit (Qiaquick PCR Purification Kit, Qiagen) removed.
  • a restriction enzyme here Nallall (Fermentas)
  • Fragments with an average size of 400 bp are generated.
  • the cleaved fragments have 3 'overhanging CATG ends and are ligated to the sequence sections with the oligomer with the genomic sequence TGTCATCCTGTTGTCATG with addition of T4-DNA ligase according to standard
  • the single-stranded ends are then supplemented to form the double-strand with Klenow enzyme (DNA polymerase I, Röche Molecular Biochemicals) and dNTPs (FIG. La).
  • Klenow enzyme DNA polymerase I, Röche Molecular Biochemicals
  • dNTPs FIG. La
  • a reference DNA proceed as follows: In the following step, the ligated sequence sections are amplified in a PCR reaction with the addition of primer oligonucleotides with the sequence TGTCATCCTGTTGTCATG and with a heat-resistant DNA polymerase. The PCR reaction is carried out in
  • the DNA is treated using bisulfite (hydrogen sulfite, disulfite) in such a way that all of the cytosines that are not methylated at the 5-position of the base are changed in such a way that a base which is different with regard to the base pairing behavior is formed, while the base in 5-position methylated cytosines remain unchanged.
  • bisulfite hydrogen sulfite, disulfite
  • the treated DNA sample is diluted with water or an aqueous solution. Desulfonation of the DNA (20 min, 96 ° C.) at pH 9 is then preferably carried out.
  • the DNA sample is amplified with the primers now complementary to the bisulfite-treated DNA, again in a polymerase chain reaction.
  • the PCR reaction is carried out in the master cycler gradient (Eppendorf, Hamburg) with the following parameters: Denaturation: 15 minutes (min) at 96 ° C, the following cycles are repeated 45 times: 60 seconds (sec) at 96 ° C , 45 sec at 42 ° C, 60 sec at 72 ° C and subsequent incubation for 10 minutes at 72 ° C (Fig. Ib).
  • the cut DNA ligated with adapters (FIG. 1 a) must be treated with bisulfite.
  • a PCR is carried out, the primer oligonucleotides with the sequences TGTTATTTTGTT-GTTTAG and TATCATCCTATTATGATA being used.
  • the PCR reaction is carried out in the master cycler gradient (Eppendorf, Hamburg) with the following parameters: Denaturation: 15 minutes (min) at 96 ° C, the following cycles are repeated 45 times: 60 seconds (sec) at 96 ° C, 45 sec at 42 ° C, 60 sec at 72 ° C and subsequent incubation for 10 minutes at 72 ° C.
  • the detection of the hybridization product is based on Cy5 fluorescence-labeled primer oligonucleotides which were used for the amplification.
  • a hybridization reaction of the amplified DNA with the oligonucleotide only occurs if there is a methylated cytosine in the bisulfite-treated DNA at this point. The methylation status of the respective cytosine to be examined thus decides on the hybridization product.
  • Figure la Restriction enzyme, Nlalll b. Adapter 1 5 '-TGTCATCCTGTTGT, ligase e.g. T4 DNA c. Klenow enzyme (DNA polymerase I), dNTP's, buffer d. Cleaning (Qiaquick cleaning kit)
  • Figure Ib e. PCR, primer 5 '-TGTCATCCTGTTGTCATG, dNTP's, buffer, TAQ f. Reaction with hydrogen sulfite g. PCR, primer 1 5 '-TGTTATTTTGTTGTTATG, primer 2 5'- TATCATCCTATTATCATA
  • Figure 2a Reaction with hydrogen sulfite b. PCR, primer 1 5 '-TGTTATTTTGTTGTTATG, primer 2 5'- TATCATCCTATTATCATA

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Abstract

L'invention concerne un procédé de préparation d'ADN déméthylé utilisé comme matière de référence pour l'analyse de méthylations par cytosine dans des échantillons d'ADN génomiques par utilisation d'amplifications complexes.
EP01989384A 2000-12-06 2001-12-05 Procede pour quantifier des methylations par cytosine dans de l'adn genomique amplifie de maniere complexe Withdrawn EP1366190A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10061348A DE10061348C2 (de) 2000-12-06 2000-12-06 Verfahren zur Quantifizierung von Cytosin-Methylierungen in komplex amplifizierter genomischer DNA
DE10061348 2000-12-06
PCT/DE2001/004617 WO2002046452A2 (fr) 2000-12-06 2001-12-05 Procede pour quantifier des methylations par cytosine dans de l'adn genomique amplifie de maniere complexe

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EP1366190A2 true EP1366190A2 (fr) 2003-12-03

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US (1) US20050019762A1 (fr)
EP (1) EP1366190A2 (fr)
AU (1) AU2002227866A1 (fr)
DE (1) DE10061348C2 (fr)
WO (1) WO2002046452A2 (fr)

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AU2002227866A1 (en) 2002-06-18
WO2002046452A3 (fr) 2003-09-18
US20050019762A1 (en) 2005-01-27
DE10061348C2 (de) 2002-10-24
DE10061348A1 (de) 2002-06-20
WO2002046452A2 (fr) 2002-06-13

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