Molecular - PCR - Methylation-specific PCR

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Molecular Pathology

Polymerase Chain Reaction

Methylation-specific PCR

Author: Rodney E. Shackelford, D.O., Ph.D. (see Reviewers page)

Revised: 8 July 2010, last major update July 2010

General

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● In mammalian cells, genes are often regulated by the addition of a –CH₃ group (methylation) to specific promoter cytosine moieties that are followed by a guanosine

● The methylation of this dinucleotide, called a “CpG island”, results in inhibition of gene transcription and hence repression of gene expression

● Interestingly, cytosine methylation is very stable and heritable, and is passed through cell division, making cytosine methylation a major mechanism for gene imprinting

● Additionally, aberrant cytosine methylation is commonly seen in malignancies, where the expression of tumor suppressor genes is lowered or completely blocked by this mechanism.

Detection

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● Cytosine methylated DNA can be distinguished from cytosine unmethylated DNA by in vitro sodium bisulfite treatment, which converts unmethylated cytosine into uracil, but does not change methylated cytosine

● Using PCR with primers that bind either the modified or unmodified CpG containing sequences can be used to look for the cytosine to uracil modification: in an unmodified promoter sequence, the cytosines would bind a primer with guanosines, but with bisulfite modification, the promoter uracils would bind a primer with thymines

● Running separate PCR reactions with each set of primers (one for methylated DNA and one for nonmethyated DNA), would easily reveal the presence or absence of cytosine methylation

● When analyzed by gel electrophoresis, followed by ethidium bromide staining and UV visualization, unmethylated DNA is detected when cytosine binds to a primer with guanosine primer and methylated DNA is detected when cytosine binds to a primer with thymine

● Although this technique is commonly used, it has disadvantages of (a) the bisulfite treatment destroys about 90% of the DNA and (b) some of the reagents are toxic

End of Molecular Pathology > Polymerase Chain Reaction > Methylation-specific PCR

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