WO2002046452A3 - Verfahren zur quantifizierung von cytosin-methylierungen in komplex amplifizierter genomischer dna - Google Patents

Verfahren zur quantifizierung von cytosin-methylierungen in komplex amplifizierter genomischer dna Download PDF

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Publication number
WO2002046452A3
WO2002046452A3 PCT/DE2001/004617 DE0104617W WO0246452A3 WO 2002046452 A3 WO2002046452 A3 WO 2002046452A3 DE 0104617 W DE0104617 W DE 0104617W WO 0246452 A3 WO0246452 A3 WO 0246452A3
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WO
WIPO (PCT)
Prior art keywords
oligonucleotides
amplified
genomic dna
cytosine
adapters
Prior art date
Application number
PCT/DE2001/004617
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English (en)
French (fr)
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WO2002046452A2 (de
Inventor
Alexander Olek
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Epigenomics Ag
Alexander Olek
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Epigenomics Ag, Alexander Olek filed Critical Epigenomics Ag
Priority to EP01989384A priority Critical patent/EP1366190A2/de
Priority to AU2002227866A priority patent/AU2002227866A1/en
Priority to US10/433,742 priority patent/US20050019762A1/en
Publication of WO2002046452A2 publication Critical patent/WO2002046452A2/de
Publication of WO2002046452A3 publication Critical patent/WO2002046452A3/de

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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

Abstract

Die vorliegende Erfindung beschreibt ein Verfahren zur Bereitstellung von demethylierter DNA als Referenzmaterial für die Analyse von Cytosin-Methylierungen in genomischen DNA-Proben unetr Verwendung komplexer Amplifikationen. Dazu werden im Einzelnen die folgenden Verfahrensschritte durchgeführt : a) Ein genomische DNA-Probe wird mit Primem amplifiziert, die entweder sehr kurze oder degenerierte Oligonukleotide oder zu Adaptoren komplementäre Oligonukleotide sind. Für den zweiten Fall wird vor der Amplifikation mit einem Restriktionsenzym geschnitten und die Adaptoren, unetr denen man kurze Nukleotidfragmente bekannter Sequenz versteht, an die Enden der entstandenen DNA Fragmente ligiert. Die Amplifikate werden chemisch derart behandelt, dass an der 5-Position unmethylierte Cytosinbasen in Uracil, Thymin oder in eine andere im Hybridisierungsverhalten dem Cytosin unähnliche Base umgewandelt werden, während die 5-Methylcytosinbasen im wesentlich unverändert bleiben. Dies wird im folgenden unetr chemischer Vorbehandlung verstanden. Die chemisch vorbehandelten Amplifikate werden wiederum amplifiziert. Als Primer werden dazu entweder mehrere spezifisch hybridisierende Oligonukleotide oder aber zu den Adaptoren komplementäre Oligonukleotide verwendet. Für den letzteren Fall wird ebenfalls die chemische Vorbehandlung durchgeführt.
PCT/DE2001/004617 2000-12-06 2001-12-05 Verfahren zur quantifizierung von cytosin-methylierungen in komplex amplifizierter genomischer dna WO2002046452A2 (de)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP01989384A EP1366190A2 (de) 2000-12-06 2001-12-05 Verfahren zur quantifizierung von cytosin-methylierungen in komplex amplifizierter genomischer dna
AU2002227866A AU2002227866A1 (en) 2000-12-06 2001-12-05 Method for quantifying cytosine methylations in genomic dna that is amplified in a complex manner
US10/433,742 US20050019762A1 (en) 2000-12-06 2001-12-05 Method for quantifying cytosine methylations in genomic dna that is amplified in a complex manner

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10061348.9 2000-12-06
DE10061348A DE10061348C2 (de) 2000-12-06 2000-12-06 Verfahren zur Quantifizierung von Cytosin-Methylierungen in komplex amplifizierter genomischer DNA

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WO2002046452A2 WO2002046452A2 (de) 2002-06-13
WO2002046452A3 true WO2002046452A3 (de) 2003-09-18

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

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Also Published As

Publication number Publication date
WO2002046452A2 (de) 2002-06-13
EP1366190A2 (de) 2003-12-03
US20050019762A1 (en) 2005-01-27
DE10061348C2 (de) 2002-10-24
AU2002227866A1 (en) 2002-06-18
DE10061348A1 (de) 2002-06-20

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