Microsoft® Word™ Macro for Analysis of Cytosine Methylation by the Bisulfite Deamination Reaction

Abstract

Cytosine methylation at CpG dinucleotides is an important control mechanism in development, differentiation, and neoplasia. Bisulfite genomic sequencing and its modifications have been developed to examine methylation at these CpG dinucleotides. To use these methods, one has to (i) manually convert the sequence to that produced by bisulfite conversion and PCR amplification, taking into account that cytosine residues at CpG dinucleotides may or may not be converted depending on their methylation status, (ii) identify relevant restriction sites that may be used for methylation analysis, and (iii) conduct similar steps with the other DNA strand since the two strands of DNA are no longer complementary after bisulfite conversion. To automate these steps, we have developed a macro that can be used with Microsoft® Word™. This macro (i) converts genomic sequence to modified sequence that would result after bisulfite treatment facilitating primer design for bisulfite genomic sequencing and methylation-sensitive PCR assay and (ii) identifies restriction sites that are preserved in bisulfite-converted and PCR-amplified product only if cytosine residues at relevant CpG dinucleotides are methylated (and thereby not converted to uracil) in the genomic DNA.