WO2007033834A2 - Procédé d'analyse de la méthylation d'acides nucléiques - Google Patents

Procédé d'analyse de la méthylation d'acides nucléiques Download PDF

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Publication number
WO2007033834A2
WO2007033834A2 PCT/EP2006/009233 EP2006009233W WO2007033834A2 WO 2007033834 A2 WO2007033834 A2 WO 2007033834A2 EP 2006009233 W EP2006009233 W EP 2006009233W WO 2007033834 A2 WO2007033834 A2 WO 2007033834A2
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WIPO (PCT)
Prior art keywords
methylation
nucleic acid
amplification reaction
methylated
primer
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PCT/EP2006/009233
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English (en)
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WO2007033834A3 (fr
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Sonja Sievers
Oliver MÜLLER
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MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Publication of WO2007033834A2 publication Critical patent/WO2007033834A2/fr
Publication of WO2007033834A3 publication Critical patent/WO2007033834A3/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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

Definitions

  • the present invention relates to a method for the analysis of nucleic acid methylation.
  • ms-SNuPE methylation-sensitive single-nucleotide primer extension
  • An object of the present invention was therefore to provide an improved method for the analysis of methylation, in particular a method which allows for fast and easy quantification of the proportion of methylated and non- methylated nucleic acids in a given sample.
  • This object is solved according to the invention by a method for the analysis of nucleic acid methylation comprising the steps:
  • a fast, easy and low cost method for the analysis in particular also for the quantitative analysis of nucleic acid methylation, in particular DNA methylation is provided which can be automated.
  • a nucleic acid to be analyzed is provided, which nucleic acid, in particular, is a methylated nucleic acid.
  • the nucleic acid is DNA and most preferably genomic DNA.
  • the nucleic acid to be analyzed preferably is a human genomic DNA which is methylated. In human cells the motif CpG is methylated.
  • an in vitro methylated nucleic acid can be used.
  • Methylation can be performed by treating the nucleic acid with an enzyme, in particular with a methyl transferase.
  • Suitable methyl transferases include M.Sssl, Dnmti human DNA (cytosine-5) methyltransferase, AIu I methylase, BamH I methylase/, dam/methylase, EcoR I methylase, Hae III methylase, Hha I methylase, Hpa Il methylase, Msp I methylase and Taq I methylase. Particularly preferred, M.Sssl is used.
  • This methyl transferase specifically methylates cytosine nucleotides within the motif CpG and is therefore suitable for methylation and analysis of human DNA.
  • Other enzymes are used for analysis of bacterial, animal or plant DNA, depending on the respective methylation motifs.
  • a next step b) specifically methylated nucleic acid or non-methylated nucleic acid are modified.
  • Modification includes conversion of methylated nucleotides or non-methylated nucleotides into other chemical entities, which lateron allow for distinction between methylation and non-methylated species.
  • all non-methylated cytosines are modified and all methylated cytosines are not converted.
  • the nucleic acid, in particular genomic DNA is modified using sodium bisulfite. Such procedure is e.g. described by M. Frommer et al., Proc. Natl. Acad. Sci. USA 89 (1992), 1827-1831. Using sodium bisulfite non-methylated cytosine nucleotides are modified to uracil nucleotides, while methylated cytosine nucleotide are not converted.
  • a first amplification reaction is performed, which amplification reaction is non-specific for methylation.
  • Non-specific for methylation means that both methylated and non-methylated nucleic acids or modified and non- modified nucleic acids, respectively, are amplified.
  • the first amplification reaction is a polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • primers are used which anneal independently from previous methylation to the DNA or DNA fragment to be analyzed, e.g. to a fragment of a genomic template.
  • a first amplification product is obtained.
  • cytosines which have not been methylated and were therefore converted to uracils are now replicated as thymine nucleotides.
  • non-methylated C e.g. non-methylated CpG pairs
  • T in particular into TpG pairs by bisulfite treatment and following the first amplification reaction, in particular a PCR.
  • the amplification product of the first amplification reaction then serves as template in a second amplification reaction in step (d) of the present invention.
  • the second amplification reaction is a solid phase amplification reaction, in particular a solid-phase PCR using a first immobilized primer, which is non- specific for methylation and a second non-immobilized methylation-specific primer.
  • the first methylation unspecific primer is covalently bound to a solid phase and thus immobilized.
  • a suitable solid phase are: a wall of a reaction tube, which can be a single tube or a well of a Multiwell plate, a magnetic or non-magnetic microbead, a complex of several macromolecules (nanobead), a carbohydrate-based or polymer-based membrane.
  • the sequence is amplified.
  • the second primer only anneals to sequences which were originally methylated or only to sequences which were originally non- methylated.
  • the second primer comprises a marker group which allows for detection and in particular for quantitative detection.
  • Suitable marker groups include e.g. a part of a specific binding pair, such as biotin, avidin or streptavidin, a direct label such as a colour label, a label which is detectable at fluorescent, UV, visible or infrared light, a radioactive label, a protein label, e.g.
  • the marker group is biotin, e.g. a biotinylated second primer is used.
  • streptavidin can be added, which binds to the immobilized biotinylated amplification product.
  • a biotinylated enzyme e.g. biotinylated horse-radish peroxidase (HRP) or another reporter enzyme is added which binds to the streptavidin.
  • Quantification can then be performed by enzymatic colorimetric detection, e.g. by adding TMB as colorless substrate, which is converted into a blue substrate in a colorimetric enzyme reaction.
  • the number of cycles in the first amplification and/or the second amplification reaction is ⁇ 50, in particular ⁇ 35 and more preferred ⁇ 30.
  • Using a low number of amplification cycles, in particular in the second amplification reaction results in that the amount of amplification product correlates linear with the amount of templates. Thus, a fast and immediate quantification can be made.
  • the second amplification reaction is performed using an additional third primer.
  • one of the second and third primer is annealing specifically to the methylated sequence, while the other is specifically annealing to non-methylating sequence.
  • Figure 1 schematically shows steps (d) and (e) of the present invention.
  • DNA is modifed with bisulfite and amplified in PCR which is unspecific for methylation (not shown).
  • the colorimetric detection is made after solid phase (PCR) binding of streptavidin and biotinylated HRP and the addition of TMB.
  • Figure 2 shows the specificity of the inventive method. Shown is the detection of the product of a solid-phase of a PCR, whereby the product is indicated by yellow color.
  • Figure 3 shows quantification of the proportion of methylated DNA. Known percentages of methylated DNA are shown. The columns indicate the absorptions of the samples. The results are mean values from two measurements. Examples
  • Genomic DNA is isolated from human embryonic kidney 293 (HEK293) cells using standard method
  • the bisulfite modification of 1 ⁇ g DNA starts with an incubation in 50 ⁇ l denaturing buffer (300 mM
  • the DNA is deaminated in 4 M sodium bisulfite and 10 mM h.' ⁇ rochinone for 4 h at 55°C.
  • the DNA is desalted using the Qiagen PCR purification kit and desulfonated in denaturins buffer at 37 0 C for 15 min.
  • Primer A 5'- GGGTTGTATTAATATAGTTATAT-3' is complementary to the sequence 829 through 851 of the promoters of the APC gene (Gene Database U02509.1).
  • Primer B 5'- TTCCTTACTTACTAAAAACTAAAA-S' is complementary to the promoter sequence from position 600 through 628.
  • the PCR is performed with 600 nM primer A, 600 nM primer B in 25 ⁇ l PCR buffer (Ix Qiagen PCR Puffer, 200 ⁇ M dNTP, 0,5 U/ml Hot Start Taq Polymerase (Qiagen)) in 30 cycles.
  • One cycle consists of incubations at 94 0 C for 30 s, at 50 0 C for 30 s and at 72 0 C for 30 s. Step 5.
  • the phosphorylated primer C S'-P-TTTTTTTTTTGGGTTGTATTAATATAGTTATAT-S' is coupled to the activated solid phase ⁇ £ a Nucleolink Strip (Nunc).
  • 100 ⁇ primer binding buffer 5 ⁇ M primer C, 10 mM methylimidazol, 10 mM l-ethy]-3 -(3 -dimethyl - aminopropyl)-carbodiimid
  • the sequence of primer C is identical to the sequence of primer A with additional 10 thymine nucleotides at the 5'-end.
  • the second PCR is performed with 500 nM primer D and 62.5 nM primer C in 50 ⁇ PCR buffer.
  • the PCR is done in 25 cycles, when one cycle consists of incubations at 94 0 C for 30 s, at 50°C for 30 s and at 72°C for 30 s.
  • the 5'-end biotinylated primer D 5'B-CGAAATACGAATCGAAAAACG-S ' is complementary to the sequence 707 to 727 of the promoter of the APC gene.
  • Primer D anneals only to DNA, which was originally non-methylated.
  • the immobilized primer C is used. Step 7. Washing and detection
  • washing buffer 100 mM Tris/HCl (pH 7.5), 150 mM NaCl, 0.1 % Tween 20
  • the well is incubated for 5 min in 200 ⁇ l washing buffer. Washing is made three times
  • 100 ⁇ detection buffer 1 100 mM Tris/HCl (pH 7.5), 150 mM Na Q , 0.1 % Tween 20, 0.1 ⁇ gl ⁇ str ⁇ ptavidin, 25 ⁇ °l ⁇ biotin-HRP
  • 100 ⁇ detection buffer 1 100 mM Tris/HCl (pH 7.5), 150 mM Na Q , 0.1 % Tween 20, 0.1 ⁇ gl ⁇ str ⁇ ptavidin, 25 ⁇ °l ⁇ biotin-HRP

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
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  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé d'analyse de la méthylation d'acides nucléiques.
PCT/EP2006/009233 2005-09-23 2006-09-22 Procédé d'analyse de la méthylation d'acides nucléiques WO2007033834A2 (fr)

Applications Claiming Priority (2)

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EP05020789.3 2005-09-23
EP05020789 2005-09-23

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WO2007033834A2 true WO2007033834A2 (fr) 2007-03-29
WO2007033834A3 WO2007033834A3 (fr) 2007-07-12

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009006543A1 (fr) * 2007-07-02 2009-01-08 Euclid Diagnostics Llc Procédés pour évaluer l'état de méthylation d'un polynucléotide

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003031649A2 (fr) * 2001-10-05 2003-04-17 Epigenomics Ag Procede de detection de la cytosine dans des ilots cpg
US20030165884A1 (en) * 1999-12-20 2003-09-04 Stemcyte, Inc. High throughput methods of HLA typing
WO2004020662A2 (fr) * 2002-08-27 2004-03-11 Epigenomics Ag Procede et acides nucleiques servant a l'analyse de troubles lies a la proliferation des cellules mammaires

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030165884A1 (en) * 1999-12-20 2003-09-04 Stemcyte, Inc. High throughput methods of HLA typing
WO2003031649A2 (fr) * 2001-10-05 2003-04-17 Epigenomics Ag Procede de detection de la cytosine dans des ilots cpg
WO2004020662A2 (fr) * 2002-08-27 2004-03-11 Epigenomics Ag Procede et acides nucleiques servant a l'analyse de troubles lies a la proliferation des cellules mammaires

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DOUGLAS DONNA B ET AL: "Hypermethylation of a small CpGuanine-rich region correlates with loss of activator protein-2alpha expression during progression of breast cancer." CANCER RESEARCH, vol. 64, no. 5, 1 March 2004 (2004-03-01), pages 1611-1620, XP002427779 ISSN: 0008-5472 *
FRAGA M F ET AL: "DNA METHYLATION: A PROFILE OF METHODS AND APPLICATIONS" BIOTECHNIQUES, INFORMA LIFE SCIENCES PUBLISHING, WESTBOROUGH, MA, US, vol. 33, no. 3, September 2002 (2002-09), pages 632,634,636-649, XP001107174 ISSN: 0736-6205 *
SIEVERS SONJA ET AL: "Hypermethylation of the APC promoter but lack of APC mutations in myxoid/round-cell liposarcoma" INTERNATIONAL JOURNAL OF CANCER, vol. 119, no. 10, November 2006 (2006-11), pages 2347-2352, XP002427780 ISSN: 0020-7136 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009006543A1 (fr) * 2007-07-02 2009-01-08 Euclid Diagnostics Llc Procédés pour évaluer l'état de méthylation d'un polynucléotide
US8361724B2 (en) 2007-07-02 2013-01-29 Euclid Diagnostics Llc Methods for evaluating the methylation status of a polynucleotide
US8658369B2 (en) 2007-07-02 2014-02-25 Euclid Diagnostics Llc Methods for evaluating the methylation status of a polynucleotide

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