WO2004013284A3 - A method to assess genomic dna methylation using high-performance liquid chromatography-electospray ionization mass spectrometry - Google Patents
A method to assess genomic dna methylation using high-performance liquid chromatography-electospray ionization mass spectrometry Download PDFInfo
- Publication number
- WO2004013284A3 WO2004013284A3 PCT/US2003/023212 US0323212W WO2004013284A3 WO 2004013284 A3 WO2004013284 A3 WO 2004013284A3 US 0323212 W US0323212 W US 0323212W WO 2004013284 A3 WO2004013284 A3 WO 2004013284A3
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- deoxycytidine
- methyl
- day
- dna
- liquid chromatography
- Prior art date
Links
- 238000000034 method Methods 0.000 title abstract 7
- 230000007067 DNA methylation Effects 0.000 title abstract 3
- 239000007788 liquid Substances 0.000 title 1
- 238000004949 mass spectrometry Methods 0.000 title 1
- LUCHPKXVUGJYGU-XLPZGREQSA-N 5-methyl-2'-deoxycytidine Chemical compound O=C1N=C(N)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 LUCHPKXVUGJYGU-XLPZGREQSA-N 0.000 abstract 7
- CKTSBUTUHBMZGZ-SHYZEUOFSA-N 2'‐deoxycytidine Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 CKTSBUTUHBMZGZ-SHYZEUOFSA-N 0.000 abstract 4
- CKTSBUTUHBMZGZ-UHFFFAOYSA-N Deoxycytidine Natural products O=C1N=C(N)C=CN1C1OC(CO)C(O)C1 CKTSBUTUHBMZGZ-UHFFFAOYSA-N 0.000 abstract 3
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 abstract 2
- 150000002500 ions Chemical class 0.000 abstract 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 abstract 2
- 102000004190 Enzymes Human genes 0.000 abstract 1
- 108090000790 Enzymes Proteins 0.000 abstract 1
- 208000035389 Ring chromosome 6 syndrome Diseases 0.000 abstract 1
- 238000004458 analytical method Methods 0.000 abstract 1
- 230000029087 digestion Effects 0.000 abstract 1
- 230000003301 hydrolyzing effect Effects 0.000 abstract 1
- 238000001819 mass spectrum Methods 0.000 abstract 1
- 238000005259 measurement Methods 0.000 abstract 1
- 238000004007 reversed phase HPLC Methods 0.000 abstract 1
- 238000002211 ultraviolet spectrum Methods 0.000 abstract 1
Classifications
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/02—Details
- H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
-
- 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
- C12Q1/6813—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]
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention provides a method for quantitative determination of 5-methyl-2'-deoxycytidine in human DNA using liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). The method comprises the steps of enzymatically hydrolyzing the DNA sample by sequential digestion with enzymes; separating the DNA hydrolyzates by reverse-phase high-performance liquid chromatography in isocratic mode wherein the four major DNA bases and 5-methyl-2'-deoxycytidine are resolved and eluted; identifying the 2'-deoxycytidine and 5-methyl-2'-deoxycytidine by combining diode array UV spectra analysis and mass spectra of chromatographic peaks. The isotopomes 15N3 2'-deoxycytidine and methyl-D3, ring-6-D1 5-methyl-2'-deoxycytidine are used as internal standards. Ions of m/z 126 and 130 are used to detect 5-methyl-2'-deoxycytidine and its isotopomer, and ions of m/z 112 and 115 are used to detect 2'-deoxycytidine and its stable isotopomer, respectively. The DNA methylation status is consequently calculated based on the amount of 5-methyl-2'-deoxycytidine per µg DNA with percent relative standard deviations (%RSD) for method precision of 7.1 (within-day) and 5.7 (day-to-day). The method of the present invention also allows the measurement of 5-methyl-2'-deoxycytidine expressed as a percentage of total deoxycytidine residues in genomic DNA with % RSD for method precision of 1.9 (within-day) and 1.7 (day-to-day). The LC/MS method for quantitative determination of genomic DNA methylation status is rapid, sensitive, selective and precise.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US40075602P | 2002-08-02 | 2002-08-02 | |
| US60/400,756 | 2002-08-02 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO2004013284A2 WO2004013284A2 (en) | 2004-02-12 |
| WO2004013284A3 true WO2004013284A3 (en) | 2004-07-15 |
Family
ID=31495872
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US2003/023212 WO2004013284A2 (en) | 2002-08-02 | 2003-07-25 | A method to assess genomic dna methylation using high-performance liquid chromatography-electospray ionization mass spectrometry |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2004013284A2 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7608394B2 (en) | 2004-03-26 | 2009-10-27 | Sequenom, Inc. | Methods and compositions for phenotype identification based on nucleic acid methylation |
| US8962247B2 (en) | 2008-09-16 | 2015-02-24 | Sequenom, Inc. | Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non invasive prenatal diagnoses |
| US9249456B2 (en) | 2004-03-26 | 2016-02-02 | Agena Bioscience, Inc. | Base specific cleavage of methylation-specific amplification products in combination with mass analysis |
| US9394565B2 (en) | 2003-09-05 | 2016-07-19 | Agena Bioscience, Inc. | Allele-specific sequence variation analysis |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100028877A1 (en) | 2006-10-18 | 2010-02-04 | Philipp Schatz | Molecule for providing a standard for the quantitative analysis of the methylations status of a nucleic acid |
| US8206927B2 (en) | 2007-01-23 | 2012-06-26 | Sequenom, Inc. | Method for accurate assessment of DNA quality after bisulfite treatment |
| US8476013B2 (en) | 2008-09-16 | 2013-07-02 | Sequenom, Inc. | Processes and compositions for methylation-based acid enrichment of fetal nucleic acid from a maternal sample useful for non-invasive prenatal diagnoses |
| ES2577017T3 (en) | 2009-12-22 | 2016-07-12 | Sequenom, Inc. | Procedures and kits to identify aneuploidy |
| WO2013131021A1 (en) | 2012-03-02 | 2013-09-06 | Sequenom Inc. | Methods and processes for non-invasive assessment of genetic variations |
| US10504613B2 (en) | 2012-12-20 | 2019-12-10 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
| US9920361B2 (en) | 2012-05-21 | 2018-03-20 | Sequenom, Inc. | Methods and compositions for analyzing nucleic acid |
| HK1206792A1 (en) | 2012-07-13 | 2016-01-15 | Sequenom, Inc. | Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non-invasive prenatal diagnoses |
| EP3597774A1 (en) | 2013-03-13 | 2020-01-22 | Sequenom, Inc. | Primers for dna methylation analysis |
| WO2015138774A1 (en) | 2014-03-13 | 2015-09-17 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
| CN106434847A (en) * | 2016-04-18 | 2017-02-22 | 北京中科唯新生物医学研究所有限公司 | Kit for detecting activity of MTHFR (5,10-methylene tetrahydrofolate reductase) |
| CN111220760A (en) * | 2020-03-11 | 2020-06-02 | 苏州大学附属第二医院 | A method for determining 5-methylcytosine, cytosine and 5-methyldeoxycytidine in plasma deoxyribonucleic acid |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6011200A (en) * | 1997-07-30 | 2000-01-04 | Yale University | Methods for altering the rate of plant development and plants obtained therefrom |
| US6214556B1 (en) * | 1997-11-27 | 2001-04-10 | Epigenomics Ag | Method for producing complex DNA methylation fingerprints |
| US6331393B1 (en) * | 1999-05-14 | 2001-12-18 | University Of Southern California | Process for high-throughput DNA methylation analysis |
| US6486384B1 (en) * | 1997-09-24 | 2002-11-26 | The Regents Of The University Of California | Methods and compositions for transformation of cereals using cultured shoot meristematic tissue |
| US6514698B1 (en) * | 1997-08-29 | 2003-02-04 | Osvaldo J. Lopez | DNA methyltransferase genotyping |
-
2003
- 2003-07-25 WO PCT/US2003/023212 patent/WO2004013284A2/en unknown
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6011200A (en) * | 1997-07-30 | 2000-01-04 | Yale University | Methods for altering the rate of plant development and plants obtained therefrom |
| US6514698B1 (en) * | 1997-08-29 | 2003-02-04 | Osvaldo J. Lopez | DNA methyltransferase genotyping |
| US6486384B1 (en) * | 1997-09-24 | 2002-11-26 | The Regents Of The University Of California | Methods and compositions for transformation of cereals using cultured shoot meristematic tissue |
| US6214556B1 (en) * | 1997-11-27 | 2001-04-10 | Epigenomics Ag | Method for producing complex DNA methylation fingerprints |
| US6331393B1 (en) * | 1999-05-14 | 2001-12-18 | University Of Southern California | Process for high-throughput DNA methylation analysis |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9394565B2 (en) | 2003-09-05 | 2016-07-19 | Agena Bioscience, Inc. | Allele-specific sequence variation analysis |
| US7608394B2 (en) | 2004-03-26 | 2009-10-27 | Sequenom, Inc. | Methods and compositions for phenotype identification based on nucleic acid methylation |
| US9249456B2 (en) | 2004-03-26 | 2016-02-02 | Agena Bioscience, Inc. | Base specific cleavage of methylation-specific amplification products in combination with mass analysis |
| US8962247B2 (en) | 2008-09-16 | 2015-02-24 | Sequenom, Inc. | Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non invasive prenatal diagnoses |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2004013284A2 (en) | 2004-02-12 |
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