Protocol abstract
Nature Protocols 2, - 1931 - 1936 (2007)
Published online: 2 August 2007 | doi:10.1038/nprot.2007.271
Subject Category: Nucleic acid based molecular biology
Methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) for quantitative measurement of DNA methylation
Mark L Gonzalgo1 & Gangning Liang2
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.
- Department of Urology, James Buchanan Brady Urological Institute, The Johns Hopkins Medical Institutions, 600 North Wolfe Street, Baltimore, Maryland 21287, USA.
- Department of Urology, USC/Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, NOR 7338, 1441 Eastlake Avenue, Los Angeles, California 90089, USA.
Correspondence to: Mark L Gonzalgo1 e-mail: gonzalgo@jhmi.edu
Correspondence to: Gangning Liang2 e-mail: gliang@usc.edu
nature-products
A-Z product listing
- 10
PCR buffer with MgCl2
(Promega) - 100% Ethanol(BDH Chemical)
- [32P]dCTP and [32P]TTP(PerkinElmer)
- Ethidium bromide(Sigma)
- Gel dryer(Bio-Rad)
- Glycogen(Roche)
