Optimizing Annealing Temperature in Bisulfite Methylation Analysis
Feb 7, 2007 methylation, research articles
Sodium bisulfite treatment of DNA is widely used by researchers to analyze the DNA methylation patterns of DNA regions of interest. With bisulfite treatment of DNA, researchers are able to quantitatively determine if a 5′ cytosine is methylated. If a cytosine (C) is methylated, bisulfite treatment will leave the cytosine untouched. If the cytosine is unmethylated, bisulfite treatment will convert the C to a uracil (U). Researchers can then sequence a region of interest after bisulfite treatment to determine the 5′ cytosines that are methylated and unmethylated.
A new paper in the January 2007 issue of BioTechniques shows that PCR bias can be reduced by optimizing the annealing temperature in PCR methylation analysis (1).
The main concern for PCR-based quantitative DNA methylation analysis is PCR bias, which is due to the fact that methylated and unmethylated DNA molecules sometimes amplify with greatly differing efficiencies.
After analysis of several variables in the PCR reaction mixture that could contribute to this variability, the authors concluded that increasing annealing temperature in the PCR reaction resulted in a higher level of methylation detected.
It is clear from the results that regardless of the primer system, there is a strong bias toward the amplification of unmethylated DNA, and increasing annealing temperature for PCR improved the amplification toward methylated DNA.
References:
1. Shen L, Guo Y, Ahmed S, Issa JJ. 2007. Optimizing annealing temperature overcomes bias in bisulfite PCR methylation analysis. BioTechniques 42(1):48-58.
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