WO2004013284A2 - Methode pour evaluer la methylation d'adn genomique au moyen de la spectrometrie de masse a ionisation par electronebulisation/chromatographie liquide haute performance - Google Patents
Methode pour evaluer la methylation d'adn genomique au moyen de la spectrometrie de masse a ionisation par electronebulisation/chromatographie liquide haute performance Download PDFInfo
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- WO2004013284A2 WO2004013284A2 PCT/US2003/023212 US0323212W WO2004013284A2 WO 2004013284 A2 WO2004013284 A2 WO 2004013284A2 US 0323212 W US0323212 W US 0323212W WO 2004013284 A2 WO2004013284 A2 WO 2004013284A2
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- deoxycytidine
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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
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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
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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]
Definitions
- DNA deoxyribonucleic acid
- DNA the primary structure of which codes for the amino acid sequence of proteins in the organism.
- DNA consists of two intertwined polynucleotide chains, each comprising a string of nucleic acid bases linked together by a sugar-phosphate linkage.
- sequence of the protein is determined by the sequence of four types of bases in the DNA: adenine(A), guanine (G) , cytosine (C), and thymine (T).
- the "expression" of a gene, resulting in the production of an encoded protein, involves transcribing the DNA of the gene into a nucleic acid intermediate called "messenger ribonucleic acid” (mRNA).
- mRNA messenger ribonucleic acid
- the mRNA has the same nucleotide base sequence as the DNA from which it is transcribed, except the mRNA contains another base, uracil (U , in place of thymine.
- Regulation of gene expression in a cell is what distinguishes one cell type from another.
- a cell-type-specific pattern of gene expression is established via complex interactions that can involve, for example, extracellular signals and tissue-specific transcription factors.
- a characteristic feature of many eukaryotic genomes is methylation of cytosine at the carbon 5' position of CpG dinucleotides. 1 Typically the methylation of cytosine occurs predominantly in CpG rich regions, the so-called 'CpG islands' that are largely localized in gene promoter regions or in the initial exons of genes. 2 DNA methylation is a fundamental mechanism for epigenetic control of gene expression and the maintenance of genomic integrity. " Therefore, evaluation of genomic DNA methylation status is critical for the study of cell growth regulation, tissue specific differentiation,
- the two most widely used methods for assessing genomic DNA methylation status are, a Southern blot technique that follows digestion with methylation-sensitive restriction endonucleases 1 ' 7"10 and a radioassay that utilizes a bacterial DNA methyltransferase to catalyze the de novo methylation of the cytosine- guanine doublet sites with a radioactive methyl donor in vitro ⁇ 1"15
- a radioassay that utilizes a bacterial DNA methyltransferase to catalyze the de novo methylation of the cytosine- guanine doublet sites with a radioactive methyl donor in vitro ⁇ 1"15
- Recently another method has been described based on methylation-sensitive endonucleases followed by single nucleotide extension with radiolabeled [ H]dCTP. Generally, these methods have wide variations in precision due to inconsistencies in the activity of methyl
- HPLC high performance liquid chromatography
- LC/MS Liquid Chromatography/Mass Spectrometry
- the present invention provides a new, on-line LC/MS method for the measurement of methylated cytosine residues in genomic DNA, including:
- step (d) identifying the products in step (d) by electro spray ionization enabled mass spectrometry
- the present LC/MS method allows the quantitative determination of genomic DNA methylation status.
- the technique relies on the quantitative hydrolysis of DNA, if necessary the complete removal of potential residual RNA, and enables the separation and identification of the DNA bases and 5-methyl-2'-deoxycitidine by ESI-MS.
- the quantity of DNA utilized in this method is relatively low and the on-line LC/MS method has a shorter run time for each sample compared to previously described methodologies.
- the present invention enables analysis of large
- genomic DNA is isolated by classical methods, e.g. phenol:chloroform:isoamyl alcohol, from sources including tissue and cells, as well as cultured cells.
- the DNA is hydrolyzed using standard methods including enzymatic digestion. Residual RNA may then be removed using RNase.
- Methylated and/or unmethylated stable cytosine base isotope-labeled compounds e.g.
- N 3 -2' -deoxycytidine and/or the custom-made methyl-D3, ring-6- Dl 5 -methyl-2' -deoxycytidine are then added as internal standards for 2'- deoxycytidine and/or 5-methyl-2' -deoxycytidine residues, respectively.
- Other stable cytosine base isotope-labeled compounds may be used provided they have the general form of deoxycytidine and methyl-deoxycytidine (see Figure 1), wherein N may be 14 N or 15 N, O may be 16 O or 18 O, C may be 13 C or 14 C and H may be 1H or 2 H, in any combination.
- isotope substitution is done on the pyrimidine ring, rather than on the pentose.
- the sugar generally separates from the pyrimidine.
- the sugar alone cannot be used for analysis because it is identical in Cyt and mCyt.
- a protocol in which a part of the sugar is analyzed may be used.
- the DNA hydrolyzates are separated by reverse-phase high performance liquid chromatography in isocratic mode or solution gradient mode, etc. Isocratic mode is preferable as it allows the same eluent to be used throughout the separation.
- Identification of 2' -deoxycytidine and 5-methyl-2'-deoxycytidine is obtained by combined diode array UV spectra analysis and mass spectra of chromatographic peaks using a mass spectrometer equipped with an electrospray ionization source. The DNA methylation status is then calculated based on the amount of 5-methyl-2'-deoxycytidine per ⁇ g of DNA.
- the LC/MS method of the present invention allows accurate measurement of the absolute amount of 5 -methyl-2 '-deoxycytidine in DNA by utilizing a newly synthesized methyl-D3, ring-6-Dl 5-methyl-2'-deoxycytidine stable isotopomer.
- the use of the isotope-labeled compound 15 N 3 2'-deoxycytidine as an internal standard for 2 '-deoxycytidine also allows the assessment of the amount of 5- methyl-2' -deoxycytidine relative to the total amount of cytosine residues. This method provides quantitative DNA methylation analysis that is more reliable and efficient than previous methods.
- the amount or degree of methylation of genomic DNA has implications in many conditions such as aging, genetic abnormalities, cancer and other disease states including atherosclerosis 41 , Angelman syndrome 42 , Duchenne muscular dystrophy 43 and ICF syndrome 44 , to name a few.
- Aberrant CpG island- hypermethylation which occurs at high frequency in tumors, can yield diagnostic information. Determining a patient's genome methylation by means of the present invention opens the way, in a cost-effective manner, for an unprecedented early warning diagnosis of many common cancers.
- DNA methylation is a good biomarker of DNA metabolism and the assessment of DNA methylation is crucial to investigate DNA metabolic pathways such as DNA synthesis and repair.
- DNA methylation analysis also provides information on genomic stability and gene expression. DNA methylation information can be used to study the interaction between nutrients and genes in determining DNA methylation.
- DNA methylation is a marker of one-carbon metabolism, a biochemical pathway that uses methyl-groups derived from the nutrient folate for several DNA metabolic reactions.
- DNA methylation is fundamental to the understanding of the molecular basis of a large number of diseases in which epigenetic control of DNA is involved such as cancer, degenerative diseases, neurodevelopmental disorders or others.
- the methods described herein will enable basic researchers to further probe the relationship between DNA methylation and disease.
- Toxicology The cost of bringing drug candidates through clinical trials that eventually fail due to toxicological problems is enormous. Thus, there is a greater need for methylation detection methods to "weed-out" drugs with toxicology problems at an early (pre-clinical) stage. Applying the methods of the present invention in a high throughput screening format will be helpful in determining if a particular drug impacts the methylation status of cells or tissues. Such screening would lower the likelihood that candidate drugs with mutagentic or epigenetic-based toxicity will proceed inappropriately to clinical trials.
- Figure 1 shows a chemical structure 2' -deoxycytidine and 5-methyl-2'- deoxycytidine, respectively.
- Cytosine base isotope-labeled compounds can be created by substituting any N with 4 N or 15 N, O may be 16 O or 18 O, C may be 13 C or 14 C and H may be H or H, in any combination.
- Figure 2 presents a typical LC/MS chromatogram of DNA digests.
- the top panel is represented the UV chromatogram obtained after complete enzymatic hydrolysis of human PBMC DNA and detected at 254 nm.
- the first peak eluting after 4.5 ⁇ 0.5 min corresponded to cytosine (Cyt) and the second peak eluting after 6.5 ⁇ 0.5 min corresponded to 5-methylcvtosine (mCyt).
- Cyt cytosine
- mCyt 5-methylcvtosine
- the middle panel is represented a typical MS chromatogram.
- Figure 4 demonstrates the correlation between DNA methylation and levels of plasma folate divided into tertiles and according by MTHFR genotype: C/C (left panel) and T/T (right panel).
- Figure 5 demonstrates a correlation between DNA methylation and levels of different co-enzymatic forms of folate in MTHFR T/T subjects. An inverse relationship was detected between DNA methylation (expressed in log-scale) and formylated tetrahydrofolate polyglutamates (formyl-THF, left panel). A positive correlation was detected between DNA methylation and methyltetrahydrofolate polyglutamates (methyl-THF, right panel).
- Figure 6 shows a simplified representation of the LC/MS method for the assessment of DNA methylation status. After the separation of DNA bases, the
- a Suplex pKb 100 analytical column (25 cm x 2.1 mm) protected by a 5 ⁇ m Suplex pKb 100 pre-column (2 cm x 2.1 mm) (Supelco, Bellefonte, PA, USA) was used.
- the HPLC system was controlled by an HP ChemStation software.
- the mass spectrometer from Bruker Daltonik (Bremen, Germany), was equipped with an electrospray ionization (ESI) source.
- the mass spectrometer, system was controlled by an Esquire-LC NTTM software version 4.0. Both software packages run on an HP Kayak XA PC under Microsoft Windows NTTM version 4.0 operating system.
- the mobile phase consisted of 7 mM ammonium acetate pH 6.7/methanol 5% (v/v) and was prepared using HPLC-grade purity water, methanol (both from J.T. Baker, Philipsburg, NJ), and ammonium acetate (Aldrich, Milwakee, WI). The mobile phase was filtered through a 0.2 ⁇ m nylon membrane filter (Alltech, Deerfield, IL) before use.
- the stable isotope-labeled compounds 15 N 3 2'-deoxycytidine and the custom-made methyl-D3, ring-6-Dl 5 -methyl-2 '-deoxycytidine were used as internal standards for 2 '-deoxycytidine and 5-methyl-2'-deoxycytidine residues, respectively.
- Quality control data showed a chemical purity of 98% for 15 N 3 2'-deoxycytidine and 95%+ for methyl-D3, ring-6-Dl 5 -methyl-2 '-deoxycytidine and a 98%+ isotopic enrichment for both compounds.
- CpGenomeTM universal enzymatically methylated human male genomic DNA was used (Intergen Company, Purchase, NY) to evaluate different amounts of 5-methyl-2'-deoxycytidine in human DNA.
- Genomic DNA was extracted from the buffy coat of human blood using a classical phenol:chloroform:isoamyl alcohol [25:24:1 (v/v/v)] protocol. Residual RNA was treated with both RNase A and Ti (Invitrogen, Carlsbad, CA) to a final concentration of 10 units/ml at 37°C for 1 hour. The DNA was re-precipitated with 7.5 M ammonium acetate (1:1/2, v/v) and ethanol 100% (1:2, v/v) and dissolved in TE buffer (lOmM tris HC1, ImM EDTA pH 8.0).
- Vitamin Bg (nmol//L) 30.38 33.85 N.S. (27.93-33.04) (30.17-37.97)
- Genomic DNA methylation status (ng mCyt/ ⁇ g DNA) according to MTHFR genotype and plasma folate levels (stratification by folate values above and below the median).
- RBCs folate forms distribution and DNA methylation status according to MTHFR C677T genotype c.
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Abstract
Cette invention se rapporte à une méthode permettant la détermination quantitative de 5-méthyl-2'-désoxycytidine dans un ADN humain en utilisant la spectrométrie de masse à ionisation par électronébulisation/chromatographie liquide (LC/ESI-MS). Cette méthode consiste à hydrolyser par voie enzymatique l'échantillon d'ADN par digestion séquentielle avec des enzymes; à séparer les hydrolysats d'ADN par chromatographie liquide haute performance en phase inverse en mode isocratique, dans lequel les quatre bases d'ADN majeures et la 5-méthyl-2'-désoxycytidine sont séparées par résolution et élection; et à identifier la 2'-désoxycytidine et la 5-méthyl-2'-désoxycytidine en combinant l'analyse des spectres U.V. par réseau de diodes et les spectres de masse des crêtes chromatographiques. Les isotopomères 15N3 2'-désoxycytidine et méthyl-D3, cycle-6-D1 5-méthyl-2'-désoxycytidine sont utilisés comme standards internes. Les ions de m/z 126 et 130 sont utilisés pour détecter la 5-méthyl-2'-désoxycytidine et son isotopomère, et les ions de m/z 112 et 115 sont utilisés pour détecter la 2'-désoxycytidine et son isotopomère stable, respectivement. L'état de méthylation de l'ADN est ensuite calculé sur la base de la quantité de 5-méthyl-2'-désoxycytidine par µg d'ADN avec des écarts standards relatifs en pour cent ( % RSD) pour la précision de la méthode de 7,1 (dans la journée) et de 5,7 (jour après jour). Cette méthode permet également de mesurer la 5-méthyl-2'-désoxycytidine exprimée sous la forme de pourcentage des résidus de désoxycytidine totale dans l'ADN génomique avec des écarts % RSD pour la précision de la méthode de 1,9 (dans la journée) et de 1,7 (jour après jour). Cette méthode LC/MS permettant la détermination quantitative de l'état de méthylation d'un ADN génomique est rapide, sensible, sélective et précise.
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US40075602P | 2002-08-02 | 2002-08-02 | |
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Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008047234A1 (fr) | 2006-10-18 | 2008-04-24 | Epigenomics Ag | Molécule pour élaborer un étalon d'analyse quantitative de l'état de méthylation d'un acide nucléique |
US8206927B2 (en) | 2007-01-23 | 2012-06-26 | Sequenom, Inc. | Method for accurate assessment of DNA quality after bisulfite treatment |
CN106434847A (zh) * | 2016-04-18 | 2017-02-22 | 北京中科唯新生物医学研究所有限公司 | 一种检测亚甲基四氢叶酸还原酶酶活性的试剂盒 |
US9605313B2 (en) | 2012-03-02 | 2017-03-28 | 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 |
US9926593B2 (en) | 2009-12-22 | 2018-03-27 | Sequenom, Inc. | Processes and kits for identifying aneuploidy |
US10612086B2 (en) | 2008-09-16 | 2020-04-07 | Sequenom, Inc. | Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non-invasive prenatal diagnoses |
CN111220760A (zh) * | 2020-03-11 | 2020-06-02 | 苏州大学附属第二医院 | 一种测定血浆脱氧核糖核酸中5-甲基胞嘧啶、胞嘧啶、5-甲基脱氧胞苷的方法 |
US10738358B2 (en) | 2008-09-16 | 2020-08-11 | Sequenom, Inc. | Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non-invasive prenatal diagnoses |
US11060145B2 (en) | 2013-03-13 | 2021-07-13 | Sequenom, Inc. | Methods and compositions for identifying presence or absence of hypermethylation or hypomethylation locus |
US11332791B2 (en) | 2012-07-13 | 2022-05-17 | Sequenom, Inc. | Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non-invasive prenatal diagnoses |
US11365447B2 (en) | 2014-03-13 | 2022-06-21 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
Families Citing this family (2)
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WO2005024068A2 (fr) | 2003-09-05 | 2005-03-17 | Sequenom, Inc. | Analyse de variations de sequences alleles specifiques |
EP2395098B1 (fr) | 2004-03-26 | 2015-07-15 | Agena Bioscience, Inc. | Division spécifique de base de produits d'amplification spécifique à la méthylation en combinaison avec une analyse de masse |
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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 |
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Cited By (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008047234A1 (fr) | 2006-10-18 | 2008-04-24 | Epigenomics Ag | Molécule pour élaborer un étalon d'analyse quantitative de l'état de méthylation d'un acide nucléique |
US8206927B2 (en) | 2007-01-23 | 2012-06-26 | Sequenom, Inc. | Method for accurate assessment of DNA quality after bisulfite treatment |
US8673571B2 (en) | 2007-01-23 | 2014-03-18 | Sequenom, Inc. | Method for accurate assessment of DNA quality after bisulfite treatment |
US10612086B2 (en) | 2008-09-16 | 2020-04-07 | Sequenom, Inc. | Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non-invasive prenatal diagnoses |
US10738358B2 (en) | 2008-09-16 | 2020-08-11 | Sequenom, Inc. | Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non-invasive prenatal diagnoses |
US11180799B2 (en) | 2009-12-22 | 2021-11-23 | Sequenom, Inc. | Processes and kits for identifying aneuploidy |
US9926593B2 (en) | 2009-12-22 | 2018-03-27 | Sequenom, Inc. | Processes and kits for identifying aneuploidy |
US9605313B2 (en) | 2012-03-02 | 2017-03-28 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
US10738359B2 (en) | 2012-03-02 | 2020-08-11 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
US11312997B2 (en) | 2012-03-02 | 2022-04-26 | 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 |
US11306354B2 (en) | 2012-05-21 | 2022-04-19 | Sequenom, Inc. | Methods and compositions for analyzing nucleic acid |
US11332791B2 (en) | 2012-07-13 | 2022-05-17 | Sequenom, Inc. | Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non-invasive prenatal diagnoses |
US11060145B2 (en) | 2013-03-13 | 2021-07-13 | Sequenom, Inc. | Methods and compositions for identifying presence or absence of hypermethylation or hypomethylation locus |
US11365447B2 (en) | 2014-03-13 | 2022-06-21 | Sequenom, Inc. | Methods and processes for non-invasive assessment of genetic variations |
CN106434847A (zh) * | 2016-04-18 | 2017-02-22 | 北京中科唯新生物医学研究所有限公司 | 一种检测亚甲基四氢叶酸还原酶酶活性的试剂盒 |
CN111220760A (zh) * | 2020-03-11 | 2020-06-02 | 苏州大学附属第二医院 | 一种测定血浆脱氧核糖核酸中5-甲基胞嘧啶、胞嘧啶、5-甲基脱氧胞苷的方法 |
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