Polymerase Chain Reaction
Author: Rodney E. Shackelford, D.O., Ph.D. (see Reviewers page)
Revised: 22 September 2012, last major update July 2010
Copyright: (c) 2008-2010, PathologyOutlines.com, Inc.
● 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 (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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