The use of multiple displacement amplified DNA as a control for methylation specific PCR, pyrosequencing, bisulfite sequencing and methylation-sensitive restriction enzyme PCR.
BACKGROUND
Genomic DNA methylation affects approximately 1% of DNA bases in humans, with the most common event being the addition of a methyl group to the cytosine residue present in the CpG (cytosine-guanine) dinucleotide. Methylation is of particular interest because of its role in gene silencing in many pathological conditions. CpG methylation can be measured using a wide range of techniques, including methylation-specific (MS) PCR, pyrosequencing (PSQ), bisulfite sequencing (BS) and methylation-sensitive restriction enzyme (MSRE) PCR. However, although it is possible to utilise these methods to measure CpG methylation, optimisation of the assays can be complicated due to the absence of suitable control DNA samples.


RESULTS
To address this problem, we have developed an approach that employs multiple displacement based whole genome amplification (WGA) with or without SssI-methylase treatment to generate CpG methylated and CpG unmethylated DNA, respectively, that come from the same source DNA.


CONCLUSIONS
Using these alternately methylated DNA samples, we have been able to develop and optimise reliable MS-PCR, PSQ, BS and MRSE-PCR assays for CpG methylation detection, which would otherwise not have been possible, or at least have been significantly more difficult.
Hughes
Simon
S
Tumour Biology Laboratory, John Vane Science Centre, Cancer Research UK Clincial Centre, Queen Mary's School of Medicine and Dentistry, UK. simon.hughes@cancer.org.uk
Jones
J Louise
JL
D000465:Algorithms; D001483:Base Sequence; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis*; D019175:DNA Methylation; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D011786:Quality Control; D012015:Reference Standards; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites / Q000494:pharmacology*
D013447:Sulfites; D004247:DNA; D004262:DNA Restriction Enzymes; C042345:hydrogen sulfite