Investigation of genomic methylation status using methylation-specific and bisulfite sequencing polymerase chain reaction.
Epigenetic modification plays a central role in the regulation of gene expression and therefore in the development of disease states. In particular, genomic methylation of cytosines within CpG dinucleotides is crucial to development, gene silencing and chromosome inactivation. Importantly, aberrant methylation profiles of various genes are associated with cancer and potentially autoimmune disease, brain-related disease, diabetes and heart disease. Various methods are available for the detection and quantification of methylation in a given sample. Most of these methods rely upon bisulfite conversion of DNA, which converts unmethylated cytosines to uracil, while methylated cytosines remain as cytosines. Methylation-specific amplification of DNA can be used to detect methylation at one or more (typically up to about 4) CpG sites by using primers specific to either methylated or unmethylated DNA. Alternatively, amplification of both methylated and unmethylated DNA followed by sequencing can be used to detect methylation status at multiple CpG sites. The following chapter provides protocols for bisulfite conversion of DNA, methylation-specific PCR and bisulfite sequencing PCR.
Carless
Melanie
M
Department of Genetics, Southwest Foundation for Biomedical Research, San Antonio, TX, USA.
D001483:Base Sequence; D004247:DNA / Q000032:analysis / Q000235:genetics; D019175:DNA Methylation; D017931:DNA Primers; D015894:Genome, Human / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites / Q000378:metabolism*
D017931:DNA Primers; D013447:Sulfites; D004247:DNA; C042345:hydrogen sulfite