Methylation Specific PCR (MSP): Nested PCR vs Unnested PCR

Abstract

Methylation-specific PCR (MSP) is a valuable technique for studying DNA methylation patterns due to its straightforward design and implementation, high sensitivity in detecting methylated DNA, and ability to analyze large sample sizes cost-effectively rapidly. However, researchers need to be cautious when working with new samples or samples stored in the freezer for an extended period. Freezing does not prevent the action of DNAase enzymes, which can lead to reduced DNA extraction yields. During MSP, DNA undergoes changes, and PCR is performed using two sets of primers that specifically target methylated and unmethylated DNA regions. The use of bisulfite conversion treatment, an essential step in methylation analysis, presents significant challenges. One challenge is that bisulfite treatment can cause DNA fragmentation, particularly when the initial DNA concentration is low. In nested MSP, where an additional round of PCR is performed, smearing may occur due to the high DNA concentrations used. Therefore, for new samples with a DNA concentration of 1-10 µg/µL, it is recommended to use the unnested MSP technique prior to bisulfite conversion treatment.