Methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) for quantitative measurement of DNA methylation

Mark L Gonzalgo, Gangning Liang

Research output: Contribution to journalArticle

50 Citations (Scopus)

Abstract

Methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) is a technique that can be used for rapid quantitation of methylation at individual CpG sites. Treatment of genomic DNA with sodium bisulfite is used to convert unmethylated Cytosine to Uracil while leaving 5-methylcytosine unaltered. Strand-specific PCR is performed to generate a DNA template for quantitative methylation analysis using Ms-SNuPE. SNuPE is then performed with oligonucleotide(s) designed to hybridize immediately upstream of the CpG site(s) being interrogated. Reaction products are electrophoresed on polyacrylamide gels for visualization and quantitation by phosphorimage analysis. The Ms-SNuPE technique is similar to other quantitative assays that use bisulfite treatment of genomic DNA to discriminate unmethylated from methylated Cytosines (i.e., COBRA, pyrosequencing). Ms-SNuPE can be used for high-throughput methylation analysis and rapid quantitation of Cytosine methylation suitable for a wide range of biological investigations, such as checking aberrant methylation changes during tumorigenesis, monitoring methylation changes induced by DNA methylation inhibitors or for measuring hemimethylation. Approximately two to four CpG sites can be interrogated in up to 40 samples by Ms-SNuPE in less than 5 h, after PCR amplification of the desired target sequence and preparation of PCR amplicons.

Original languageEnglish (US)
Pages (from-to)1931-1936
Number of pages6
JournalNature Protocols
Volume2
Issue number8
DOIs
StatePublished - Aug 2007
Externally publishedYes

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Methylation
DNA Methylation
Nucleotides
Cytosine
Polymerase Chain Reaction
DNA
5-Methylcytosine
Uracil
Reaction products
Oligonucleotides
Amplification
Assays
Carcinogenesis
Visualization
Throughput

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) for quantitative measurement of DNA methylation. / Gonzalgo, Mark L; Liang, Gangning.

In: Nature Protocols, Vol. 2, No. 8, 08.2007, p. 1931-1936.

Research output: Contribution to journalArticle

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