DNA methylation analysis by bisulphite conversion is a versatile method that facilitates the identification and quantification of DNA methylation at single nucleotide resolution. However, bisulphite conversion relies on full conversion with every unmethylated cytosine being deaminated to uracil and only methylated cytosines remaining unreactive.
The problem arises when the conversion is incomplete, in which unconverted unmethylated cytosines may be incorrectly interpreted as methylated cytosines.
Two types of bisulfite-conversion error may occur—inappropriate conversion of 5-methylcytosine to thymine and failure to convert unmethylated cytosine to uracil.
Other disadvantages include:
- Bisulfite treatment requires hours of exposure and, sometimes, thermal denaturation.
- The concentration of DNA and its quality can also affect the efficiency of the bisulphite reaction and ultimate PCR yield.
- DNA degradation of the bisulphite conversion protocol can occur.
- A larger amount of input DNA is required to have sufficient material for downstream applications.
However, formalin-fixed, paraffin-embedded (FFPE) and degraded DNA samples can be effectively bisulphite converted and amplified using this protocol.
