How bisulfite treatment coupled with PCR enable the identification of DNA methylation? Bisulfite conversion is regarded as the gold standard in defining DNA methylation status. Treatment of DNA with sodium bisulfite converts unmethylated cytosine to
How bisulfite treatment coupled with PCR enable the identification of DNA methylation?
Bisulfite conversion is regarded as the gold standard in defining DNA methylation status. Treatment of DNA with sodium bisulfite converts unmethylated cytosine to uracil, which is subsequently converted to thymine during PCR amplification, while methylcytosine remains unchanged (Fig.
How do you calculate bisulfite conversion rate?
The bisulfite conversion rate of each base (non-CpG) can be calculated as T / (T + C) * 100, where T is thymine and C is cytosine read numbers on that base. Below is from the aligned (by bismark) and methylation-called (by methylKit) file of one sample (CHH context).
How is the quality of DNA used in bisulfite conversion?
The quality of the DNA, in terms of size, used as input for bisulfite conversion is the most important factor when assessing recovery. Degraded starting material will lead to increased sample loss during the bisulfite conversion process.
Can a bisulfite PCR be used for direct sequencing?
Direct sequencing of bisulfite PCR products is not recommended as it generally yields poor sequencing quality and will confound quantification of partially methylated sites. Pyrosequencing can be used as an alternative to direct sequencing.
When to use bisulfite primer for methylation analysis?
Bisulfite PCR is the most common technique used for methylation analysis of bisulfite-converted DNA and also the most susceptible to mishap. Keep in mind the DNA will be significantly fragmented, as the strands are no longer complementary, and almost completely devoid of cytosine. Primer design is the key to successful bisulfite PCR.
How does sodium bisulfite work on methylated cytosines?
Sodium bisulfite deaminates non-methylated cytosines to uracil and leaves methylated cytosines untouched (in other words methylated cytosines are resistant to modification induced by sodium bisulfite).