Profiling DNA methylation from small amounts of genomic DNA starting material: efficient sodium bisulfite conversion and subsequent whole-genome amplification.
noindent Sodium bisulfite modification-based fine mapping of methylated cytosines represents the gold standard technique for DNA methylation studies. A major problem with this approach, however, is that it results in considerable DNA degradation, and large quantities of genomic DNA material are needed if numerous genomic regions are to be profiled. This chapter describes a method for profiling DNA methylation from small amounts of genomic-DNA starting material utilizing an efficient sodium bisulfite conversion method followed by whole-genome amplification (WGA). WGA is a useful method to overcome the problem of low initial amount of DNA and/or severe DNA degradation during conventional sodium bisulfite treatment in studies investigating DNA methylation. WGA is a relatively inexpensive process that can be optimized for high-throughput application and enables the thorough investigation of methylation at numerous genomic locations on samples for which DNA availability is low. Data from our lab has demonstrated that bisulfite-treated DNA amplified using WGA can be used for a range of downstream DNA methylation mapping procedures, including bisulfite-primer optimization, the sequencing of cloned PCR products, MS-SNuPE, and Pyrosequencing.
Mill
Jonathan
J
The Krembil Family Epigenetics Laboratory, Centre for Addiction and Mental Health, Toronto, Ontario, Canada.
Petronis
Arturas
A
D018899:CpG Islands; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016172:DNA Fingerprinting / Q000379:methods*; D019175:DNA Methylation; D017931:DNA Primers; D016678:Genome; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction / Q000379:methods; D013447:Sulfites
D017931:DNA Primers; D013447:Sulfites; D004247:DNA; C009279:sodium bisulfite