Bisulfite Conversion

Bisulfite Conversion is a process in which genomic DNA is denatured (made single-stranded) and treated with sodium bisulfite, leading to deamination of unmethylated cytosines into uracils, while methylated cytosines (both 5-methylcytosine and 5-hydroxymethylcytosine) remain unchanged. The DNA is then amplified by PCR where the uracils are converted to thymines. Bisulfite converted DNA can be analyzed for gene-or allele-specific methylation patterns with primers that differentiate between methylated and unmethylated sequences, or adapted for genome-wide DNA methylation analysis. Comparing the sequence of the converted DNA to untreated DNA creates a methylation profile of the sample. The results provide single nucleotide resolution information about the methylation status.

Figure 1: DNA sequence following bisulfite conversion and PCR amplification.

The original DNA sequence contains six cytosine residues in which 3 are methylated (Me) and 3 are unmethylated. Following bisulfite conversion the unmethylated cytosines are converted into uracil while the methylated cytosines remain as cytosine. The methylation profile of the DNA can then be determined by PCR amplification followed by DNA sequencing, where the uracils will be detected as thymine.

Active Motif's Bisulfite Conversion Kit simplifies analysis of DNA methylation with a rapid 1.5 hour conversion reaction. The Bislfite Conversion Kit comes complete with optimized reagents for performing DNA conversion with bisulfite, plus time-saving DNA purification columns and positive control PCR primers that only anneal to converted human and mouse DNA to validate your results.

The FFPE Bisulfite Conversion Kit is designed to work with formalin- or paraformalin-fixed, parafin-embedded (FFPE) tissue sections or unsectioned core samples. This kit offers an optimized workflow starting with DNA isolation from FFPE sections, DNA purification, bisulfite conversion, desulfonation and on-column DNA purification to improve the recovery of high quality DNA, minimize degradation and provide greater than 99% conversion efficiency.

Additionally, for those researchers interested in Bisulfite Sequencing Services, Active Motif now offers bisulfite sequencing as a custom service. The sequencing service includes primer design, isolation of DNA from cells, bisulfite conversion of DNA, PCR amplification and gel analysis of PCR products, gel purification of PCR products, cloning of PCR products, amplification of cloned inserts, sequencing and data analysis. Click to learn more about our Bisulfite Sequencing Services.