CA2399281A1 - Method for detecting cytosine methylation in dna samples - Google Patents
Method for detecting cytosine methylation in dna samples Download PDFInfo
- Publication number
- CA2399281A1 CA2399281A1 CA002399281A CA2399281A CA2399281A1 CA 2399281 A1 CA2399281 A1 CA 2399281A1 CA 002399281 A CA002399281 A CA 002399281A CA 2399281 A CA2399281 A CA 2399281A CA 2399281 A1 CA2399281 A1 CA 2399281A1
- Authority
- CA
- Canada
- Prior art keywords
- recited
- dna
- type
- oligonucleotides
- 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.)
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Classifications
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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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
Abstract
The invention relates to a method for detecting 5-methylcytosine in genomic DNA samples. Firstly, a genomic DNA from a DNA sample is chemically reacted with a reagent, whereby 5-methylcytosine and cytosine react differently. Afterwards, the pretreated DNA is amplified while using a polymerase and at least one primer. In the next step, the amplified genomic DNA is hybridized to at least one oligonucleotide while forming a duplex, and the same is lengthened by at least one nucleotide, whereby the nucleotide carries a detectable tagging, and the lengthening is subject to the methylation status of the respective cytosine in the genomic DNA sample. In the following step, the lengthened oligonucleotides are examined for the presence of the tagging .
Claims (29)
1. A method for detecting 5-methylcytosine in genomic DNA samples, characterized in that the following steps are carried out:
(a) a genomic DNA from a DNA sample is chemically converted with a reagent, 5-methylcytosine and cyto-sine reacting differently, thus exhibiting different base pairing behaviors in the DNA duplex subsequent to the reaction;
(b) the pretreated DNA is amplified using a poly-merase and at least one oligonucleotide (type A) as a primer;
(c) the amplified genomic DNA is hybridized to at least one oligonucleotide (type B), forming a duplex, said hybridized oligonucleotides of type B, with their 3'-ends, immediately or at a distance of up to bases, adjoining the positions to be analyzed with regard to their methylation in the genomic DNA sam-ple;
(d) the oligonucleotide (type B) having a known se-quence of n nucleotides is elongated by means of a polymerase by at least one nucleotide, the nucleotide carrying a detectable label, and the elongation de-pending on the methylation status of the specific cy-tosine in the genomic DNA sample;
(e) the elongated oligonucleotides are analyzed for the presence of the label.
(a) a genomic DNA from a DNA sample is chemically converted with a reagent, 5-methylcytosine and cyto-sine reacting differently, thus exhibiting different base pairing behaviors in the DNA duplex subsequent to the reaction;
(b) the pretreated DNA is amplified using a poly-merase and at least one oligonucleotide (type A) as a primer;
(c) the amplified genomic DNA is hybridized to at least one oligonucleotide (type B), forming a duplex, said hybridized oligonucleotides of type B, with their 3'-ends, immediately or at a distance of up to bases, adjoining the positions to be analyzed with regard to their methylation in the genomic DNA sam-ple;
(d) the oligonucleotide (type B) having a known se-quence of n nucleotides is elongated by means of a polymerase by at least one nucleotide, the nucleotide carrying a detectable label, and the elongation de-pending on the methylation status of the specific cy-tosine in the genomic DNA sample;
(e) the elongated oligonucleotides are analyzed for the presence of the label.
2. The method as recited in Claim 1, characterized in that the oligonucleotides (type B) are bonded to a solid phase at defined locations.
3. The method as recited in Claim 1, characterized in that the amplificates are bonded to a solid phase at defined locations.
4. The method as recited in Claim 2, characterized in that different oligonucleotide sequences are arranged on a plane solid phase in the form of a rectangular or hexagonal lattice.
5. The method as recited in Claim 9, characterized in that the labels attached to the elongated oligonu-cleotides are identifiable at each position of the solid phase at which an oligonucleotide sequence is located.
6. The method as recited in Claim 1, characterized in that at least one primer (type A) is bonded to a solid phase during amplification.
7. The method as recited in Claim 1, 3, or 6, character-ized in that different amplificates are arranged on the solid phase in the form of a rectangular or hex-agonal lattice.
8. The method as recited in one of the preceding Claims, characterized in that, prior to the amplification, the DNA is treated with a bisulfite solution (=disulfite, hydrogen sulfite).
9. The method as recited in one of the preceding Claims, characterized in that the amplification is carried out by means of the polymerase chain reaction (PCR).
10. The method as recited in one of the preceding Claims, characterized in that the oligonucleotides of type A
used either contain only the bases T, A and C or else the bases T, A and G.
used either contain only the bases T, A and C or else the bases T, A and G.
11. The method as recited in one of the preceding Claims, characterized in that the oligonucleotides of type B
used either contain only the bases T, A and C or else the bases T, A and G.
used either contain only the bases T, A and C or else the bases T, A and G.
12. The method as recited in one of the preceding Claims, characterized in that the labels of the nucleotides are fluorescence labels.
13. The method as recited in one of the Claims 1 through 10, characterized in that the labels of the nucleo-tides are radionuclides.
14. The method as recited in one of the Claims 1 through 10, characterized in that the labels of the nucleo-tides are detachable mass labels which are detected in a mass spectrometer.
15. The method as recited in one of the Claims 1 through 10, characterized in that the elongated oligonucleo-tides altogether are detected in the mass spectrome-ter, thus being uniquely labeled by their masses.
16. The method as recited in one of the Claims 1 through 10, characterized in that in each case one fragment of the elongated oligonucleotides is detected in the mass spectrometer.
17. The method as recited in Claim 15, characterized in that the fragment of the elongated oligonucleotide is produced by digestion with one or several exo- or en-donucleases.
18. The method as recited in Claim 16, characterized in that the produced fragments have a single positive or negative net charge for better detectability in the mass spectrometer.
19. The method as recited in one of the preceding Claims, characterized in that the detection of the elongated oligonucleotides is carried out and visualized by means of matrix assisted laser desorption/ionization mass spectrometry (MALDI) or using electron spray mass spectrometry (ESI).
20. The method as recited in one of the preceding Claims, wherein the polymerases are heat-resistant DNA-polymerases.
21. The method as recited in one of the preceding Claims, wherein SNPs are also detected and visualized in ad-dition to the DNA methylation.
22. The method as recited in one of the preceding Claims, wherein the used nucleotides are terminating (type C2) and/or chain-elongating nucleotides (type C1).
23. The method as recited in Claim 22, wherein the chain-terminating nucleotide (type C2) is selected from a group comprising either the bases T and C or else the basis G and A.
24. The method as recited in Claim 22 or 23, wherein the chain-elongating nucleotides (type C1) are selected from a group comprising either the nucleobases A, T
and C or else the bases G and A and T.
and C or else the bases G and A and T.
25. The method as recited in one of the preceding Claims, characterized in that the amplification of several DNA segments is carried out in one reaction vessel.
26. The method as recited in Claim 24, characterized in that the fluorescently labeled dCTP-derivate is Cy3-dCTP or Cy5-dCTP.
27. The method as recited in Claim 2 or 3, characterized in that solid phase surface is composed of silicon, glass, polystyrene, aluminum, steel, iron, copper, nickel, silver, or gold.
28. The method as recited in Claim 1, wherein the genomic DNA is obtained from a DNA sample, sources of DNA
comprising, e.g., cell lines, blood, sputum, stool, urine, cerebral-spinal fluid, tissue embedded in par-affin, histologic object slides, and all possible combinations thereof.
comprising, e.g., cell lines, blood, sputum, stool, urine, cerebral-spinal fluid, tissue embedded in par-affin, histologic object slides, and all possible combinations thereof.
29. The method as recited in one of the preceding Claims, characterized in that methylation analyses of the up-per and lower DNA strands are carried out.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE10010280A DE10010280B4 (en) | 2000-02-25 | 2000-02-25 | Method for the detection of cytosine methylation in DNA samples |
| DE10010280.8 | 2000-02-25 | ||
| PCT/DE2001/000750 WO2001062064A2 (en) | 2000-02-25 | 2001-02-23 | Method for detecting cytosine methylation in dna samples |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2399281A1 true CA2399281A1 (en) | 2001-08-30 |
| CA2399281C CA2399281C (en) | 2012-01-24 |
Family
ID=7633311
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2399281A Expired - Fee Related CA2399281C (en) | 2000-02-25 | 2001-02-23 | Method for detecting cytosine methylation in dna samples |
Country Status (9)
| Country | Link |
|---|---|
| US (1) | US7524629B2 (en) |
| EP (1) | EP1261741B1 (en) |
| JP (1) | JP4498657B2 (en) |
| AT (1) | ATE395434T1 (en) |
| AU (2) | AU2001242280B2 (en) |
| CA (1) | CA2399281C (en) |
| DE (2) | DE10010280B4 (en) |
| ES (1) | ES2307597T3 (en) |
| WO (1) | WO2001062064A2 (en) |
Families Citing this family (36)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7582420B2 (en) | 2001-07-12 | 2009-09-01 | Illumina, Inc. | Multiplex nucleic acid reactions |
| US8076063B2 (en) | 2000-02-07 | 2011-12-13 | Illumina, Inc. | Multiplexed methylation detection methods |
| US7955794B2 (en) | 2000-09-21 | 2011-06-07 | Illumina, Inc. | Multiplex nucleic acid reactions |
| DE10010282B4 (en) * | 2000-02-25 | 2006-11-16 | Epigenomics Ag | Method for the detection of cytosine methylation in DNA samples |
| DE10010281B4 (en) * | 2000-02-25 | 2005-03-10 | Epigenomics Ag | Ligase / polymerase method for the detection of cytosine methylation in DNA samples |
| DE10151055B4 (en) * | 2001-10-05 | 2005-05-25 | Epigenomics Ag | Method for detecting cytosine methylation in CpG islands |
| US20110151438A9 (en) | 2001-11-19 | 2011-06-23 | Affymetrix, Inc. | Methods of Analysis of Methylation |
| DE10160983B4 (en) * | 2001-12-05 | 2004-12-09 | Epigenomics Ag | Method and integrated device for the detection of cytosine methylation |
| US6960436B2 (en) * | 2002-02-06 | 2005-11-01 | Epigenomics Ag | Quantitative methylation detection in DNA samples |
| US7238518B2 (en) | 2002-10-04 | 2007-07-03 | Nisshinbo Industries, Inc. | Oligonucleotide-immobilized substrate for detecting methylation |
| AU2003290223A1 (en) * | 2002-12-02 | 2004-06-23 | Solexa Limited | Determination of methylation of nucleic acid sequences |
| US20050009059A1 (en) * | 2003-05-07 | 2005-01-13 | Affymetrix, Inc. | Analysis of methylation status using oligonucleotide arrays |
| US20060134650A1 (en) * | 2004-12-21 | 2006-06-22 | Illumina, Inc. | Methylation-sensitive restriction enzyme endonuclease method of whole genome methylation analysis |
| WO2006103111A2 (en) | 2005-04-01 | 2006-10-05 | Epigenomics Ag | Improved bisulfite conversion of dna |
| EP1871912B1 (en) | 2005-04-15 | 2012-02-29 | Epigenomics AG | Method for determining DNA methylation in blood or urine samples |
| US20060292585A1 (en) * | 2005-06-24 | 2006-12-28 | Affymetrix, Inc. | Analysis of methylation using nucleic acid arrays |
| EP1969139A1 (en) | 2005-11-17 | 2008-09-17 | Epigenomics AG | Method for the determination of the dna methylation level of a cpg position in identical cells within a tissue sample |
| US7820385B2 (en) * | 2006-03-22 | 2010-10-26 | The United States Of America As Represented By The Department Of Health And Human Services, Centers For Disease Control And Prevention | Method for retaining methylation pattern in globally amplified DNA |
| US7901882B2 (en) | 2006-03-31 | 2011-03-08 | Affymetrix, Inc. | Analysis of methylation using nucleic acid arrays |
| US8084734B2 (en) * | 2006-05-26 | 2011-12-27 | The George Washington University | Laser desorption ionization and peptide sequencing on laser induced silicon microcolumn arrays |
| WO2008096146A1 (en) * | 2007-02-07 | 2008-08-14 | Solexa Limited | Preparation of templates for methylation analysis |
| US20080213870A1 (en) * | 2007-03-01 | 2008-09-04 | Sean Wuxiong Cao | Methods for obtaining modified DNA from a biological specimen |
| EP2479289B1 (en) | 2007-06-08 | 2016-04-06 | Epigenomics AG | Method for methylation analysis |
| CA2965207C (en) | 2008-08-15 | 2020-12-15 | Cascade Biosystems, Inc. | Methods using enzymatic amplification cascades for detecting target nucleic acid in a sample |
| EP2340314B8 (en) | 2008-10-22 | 2015-02-18 | Illumina, Inc. | Preservation of information related to genomic dna methylation |
| US8110796B2 (en) | 2009-01-17 | 2012-02-07 | The George Washington University | Nanophotonic production, modulation and switching of ions by silicon microcolumn arrays |
| US9490113B2 (en) * | 2009-04-07 | 2016-11-08 | The George Washington University | Tailored nanopost arrays (NAPA) for laser desorption ionization in mass spectrometry |
| ES2534200T3 (en) | 2009-08-03 | 2015-04-20 | Epigenomics Ag | Methods for preserving the complexity of the genomic DNA sequence |
| US20110104695A1 (en) | 2009-11-05 | 2011-05-05 | Epigenomics Ag | Methods of predicting therapeutic efficacy of cancer therapy |
| WO2011100749A2 (en) | 2010-02-15 | 2011-08-18 | Cascade Biosystems, Inc. | Methods and materials for detecting viral or microbial infections |
| EP2536847B1 (en) * | 2010-02-15 | 2018-08-08 | Cascade Biosystems, Inc. | Methods and materials for assessing rna expression |
| CA2790006C (en) | 2010-02-15 | 2019-09-24 | Cascade Biosystems, Inc. | Enzymatic amplification methods and materials for detecting contaminatedfood products |
| US8551701B2 (en) | 2010-02-15 | 2013-10-08 | Cascade Biosystems, Inc. | Methods and materials for detecting genetic or epigenetic elements |
| US9605312B2 (en) | 2011-12-06 | 2017-03-28 | Mdxhealth, Sa | Methods of detecting mutations in BRAF and epigenetic changes |
| EP2971169A4 (en) | 2013-03-13 | 2016-10-26 | Abbott Molecular Inc | Systems and methods for isolating nucleic acids |
| JP2016512437A (en) | 2013-03-14 | 2016-04-28 | アボツト・モレキユラー・インコーポレイテツド | Multiple methylation specific amplification systems and methods |
Family Cites Families (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6013431A (en) * | 1990-02-16 | 2000-01-11 | Molecular Tool, Inc. | Method for determining specific nucleotide variations by primer extension in the presence of mixture of labeled nucleotides and terminators |
| US5605798A (en) | 1993-01-07 | 1997-02-25 | Sequenom, Inc. | DNA diagnostic based on mass spectrometry |
| SE501439C2 (en) | 1993-06-22 | 1995-02-13 | Pharmacia Lkb Biotech | Method and apparatus for analyzing polynucleotide sequences |
| DE19515552A1 (en) * | 1995-04-27 | 1996-10-31 | Europ Lab Molekularbiolog | Simultaneous sequencing of nucleic acids |
| US5728526A (en) * | 1995-06-07 | 1998-03-17 | Oncor, Inc. | Method for analyzing a nucleotide sequence |
| EP3034626A1 (en) * | 1997-04-01 | 2016-06-22 | Illumina Cambridge Limited | Method of nucleic acid sequencing |
| AU7829398A (en) * | 1997-06-09 | 1998-12-30 | University Of Southern California | A cancer diagnostic method based upon dna methylation differences |
| DE19754482A1 (en) * | 1997-11-27 | 1999-07-01 | Epigenomics Gmbh | Process for making complex DNA methylation fingerprints |
| AUPP312998A0 (en) | 1998-04-23 | 1998-05-14 | Commonwealth Scientific And Industrial Research Organisation | Diagnostic assay |
| AU3984699A (en) | 1998-05-12 | 1999-11-29 | Whitehead Institute For Biomedical Research | Multiplex dna amplification using chimeric primers |
| WO1999067414A1 (en) * | 1998-06-24 | 1999-12-29 | Glaxo Group Limited | Nucleotide detection method |
| DE10010281B4 (en) * | 2000-02-25 | 2005-03-10 | Epigenomics Ag | Ligase / polymerase method for the detection of cytosine methylation in DNA samples |
| DE10010282B4 (en) * | 2000-02-25 | 2006-11-16 | Epigenomics Ag | Method for the detection of cytosine methylation in DNA samples |
-
2000
- 2000-02-25 DE DE10010280A patent/DE10010280B4/en not_active Expired - Fee Related
-
2001
- 2001-02-23 WO PCT/DE2001/000750 patent/WO2001062064A2/en active IP Right Grant
- 2001-02-23 AU AU2001242280A patent/AU2001242280B2/en not_active Ceased
- 2001-02-23 ES ES01915054T patent/ES2307597T3/en not_active Expired - Lifetime
- 2001-02-23 JP JP2001561142A patent/JP4498657B2/en not_active Expired - Fee Related
- 2001-02-23 AU AU4228001A patent/AU4228001A/en active Pending
- 2001-02-23 EP EP01915054A patent/EP1261741B1/en not_active Expired - Lifetime
- 2001-02-23 AT AT01915054T patent/ATE395434T1/en not_active IP Right Cessation
- 2001-02-23 US US10/220,090 patent/US7524629B2/en not_active Expired - Fee Related
- 2001-02-23 DE DE50113970T patent/DE50113970D1/en not_active Expired - Lifetime
- 2001-02-23 CA CA2399281A patent/CA2399281C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| DE10010280A1 (en) | 2001-09-06 |
| WO2001062064A3 (en) | 2002-10-03 |
| DE50113970D1 (en) | 2008-06-26 |
| WO2001062064A2 (en) | 2001-08-30 |
| EP1261741A2 (en) | 2002-12-04 |
| ATE395434T1 (en) | 2008-05-15 |
| US20030129620A1 (en) | 2003-07-10 |
| AU4228001A (en) | 2001-09-03 |
| DE10010280B4 (en) | 2006-08-10 |
| EP1261741B1 (en) | 2008-05-14 |
| CA2399281C (en) | 2012-01-24 |
| JP4498657B2 (en) | 2010-07-07 |
| US7524629B2 (en) | 2009-04-28 |
| AU2001242280B2 (en) | 2007-03-01 |
| ES2307597T3 (en) | 2008-12-01 |
| JP2003523211A (en) | 2003-08-05 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| MKLA | Lapsed |
Effective date: 20180223 |