Methods (molecular, IHC, frozen)
PCR methylation specific

Topic Completed: 1 July 2010

Minor changes: 8 July 2020

Copyright: 2008-2018,, Inc.

PubMed Search: Methylation specific PCR[TI]

Rodney E. Shackelford, D.O., Ph.D.
Page views in 2020: 41
Table of Contents
Definition / general | Detection
Cite this page: Shackelford R. PCR methylation specific. website. Accessed January 18th, 2021.
Definition / general
  • 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
  • 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:
    1. Bisulfite treatment destroys about 90% of the DNA
    2. Some of the reagents are toxic
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