Biomedical Engineering Reference
In-Depth Information
4
Qualitative and Quantitative Polymerase Chain
Reaction-Based Methods for DNA Methylation Analyses
Ivy H. N. Wong
Summary
DNA methylation can be analyzed easily by qualitative or quantitative polymerase
chain reaction (PCR)-based methods, including methylation-specific PCR (MSP),
bisulfite sequencing, methylation-sensitive restriction enzyme PCR, combined bisulfite
restriction analysis (COBRA), methylation-sensitive single nucleotide primer extension
(Ms-SNuPE), and quantitative real-time MSP. MSP, which couples the bisulfite modifi-
cation of DNA and PCR, is fast, highly sensitive, specific, and widely applied for DNA
methylation analyses. Bisulfite modification converts unmethylated cytosine to uracil,
whereas methylcytosine remains unmodified. Most of these methods require specific
PCR primers that are designed to distinguish between methylated and unmethylated DNA
sequences. Bisulfite
sequencing is comparatively time-consuming. Methylation-sensitive
restriction enzyme PCR combines methylation-sensitive restriction enzyme digestion
and PCR. After enzyme digestion, PCR products are obtained if the enzyme does not
digest the methylated CpG sites within the specified DNA region. COBRA, Ms-SNuPE,
and quantitative real-time MSP allow the quantitative analyses of DNA methylation.
Key Words:
DNA methylation; methylation-specific PCR (MSP); bisulfite sequenc-
ing; methylation-sensitive restriction enzyme PCR; combined bisulfite restriction analy-
sis (COBRA); methylation-sensitive single nucleotide primer extension (Ms-SNuPE);
quantitative real-time MSP.
1. Introduction
DNA methylation takes place after DNA synthesis by the enzymatic trans-
fer of a methyl group from the methyl donor
S
-adenosylmethionine to the car-
bon-5 position of cytosine. Cytosines (Cs) usually located 5' to guanosines
(Gs) are differentially methylated in the human genome
(
1
)
. Non-CpG-rich
sequences are interspersed with CpG islands, which are approx 500 bp long
with G to C contents 55% and observed over expected frequencies of CpG
