Screening for and analysis of methylation differences using methylation-sensitive single-strand conformation analysis.
Methylation-sensitive single-strand conformation analysis (MS-SSCA) is a method of screening for methylation changes at CpG sites in a region of DNA. After bisulfite modification, the region of interest is amplified using primers specific for bisulfite-modified sequences. The amplified products are denatured and run on a nondenaturing polyacrylamide gel. The sequence differences caused by methylation lead to the formation of different secondary structures (conformers) with different mobilities. MS-SSCA is a convenient and rapid method for screening large numbers of samples for methylation. Individual bands can readily be isolated and sequenced allowing more detailed analysis of methylation changes. In this article, we present a protocol for MS-SSCA and outline strategies for the design of primers for amplifying bisulfite-modified DNA sequences.
Dobrovic
Alexander
A
Department of Haematology-Oncology, University of Adelaide, Adelaide, Australia. a.dobrovic@pmci.unimelb.edu.au
Bianco
Tina
T
Tan
Lor Wai
LW
Sanders
Tanya
T
Hussey
Damian
D
D019175:DNA Methylation; D017931:DNA Primers; D016133:Polymerase Chain Reaction / Q000379:methods; D018807:Polymorphism, Single-Stranded Conformational; D013447:Sulfites / Q000378:metabolism
D017931:DNA Primers; D013447:Sulfites