Is bisulfite-converted DNA single-stranded?
Is bisulfite-converted DNA single-stranded?
Uracil is then converted to thymine upon PCR amplification. Note that bisulfite-converted DNA is a single-stranded DNA because the two strands of DNA are no longer complementary after conversion.
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).
What is the principle of Methylation-Specific PCR?
Methylation-specific PCR (MS-PCR or MSP) is one of the most commonly used methods for gene/sequence-specific detection of DNA methylation. The DNA undergoes bisulfite conversion of cytosine to uracil and then the methylated sequences are selectively amplified with primers specific for methylation.
Does DNA methylation affect PCR?
Recent studies show that cytosine methylation alters DNA mechanical properties and suggest that DNA methylation may directly or indirectly influence the effectiveness of DNA amplification during PCR.
What is DNA methylation test?
Methylation analysis is the study of chromosomal patterns of DNA or histone modification by methyl groups. Methylation constitutes an epigenetic mark because its location can affect the regulation of gene expression in a heritable fashion.
What happens if the conversion of bisulfite is incomplete?
Incomplete conversion. Bisulfite sequencing relies on the conversion of every single unmethylated cytosine residue to uracil. If conversion is incomplete, the subsequent analysis will incorrectly interpret the unconverted unmethylated cytosines as methylated cytosines, resulting in false positive results for methylation.
How is the conversion of bisulfite to uracil performed?
Bisulfite conversion involves the deamination of unmodified cytosines to uracil, leaving the modified bases 5-mC and 5-hmC. This procedure can then be followed either by PCR amplification or massively parallel sequencing methods to reveal the methylation status of every cytosine in gene specific amplification or whole genome amplification.
Why is the bisulfite conversion of DNA important?
Bisulfite conversion of DNA is an essential first step for many gene-specific and epigenome-wide analyses. Since current sequencing methods cannot distinguish between methylated and unmethylated cytosines, different methylation-specific sequences must be created.
What is the oxidative method for bisulfite conversion?
Oxidative bisulfite (oxBS) method was developed by the Balasubramanian and Reik laboratories [30]. This method allows discriminating 5hmC from 5mC during bisulfite conversion by selectively oxidizing 5hmC to 5fC (and 5caC to some extent) with potassium perruthanate (KRuO 4 ).
