methBLAST is a sequence similarity search program designed to explore in silico bisulfite modified DNA (either or not methylated at its CpG dinucleotides). The tool is mainly developed to find primer binding sites and hence addresses specificity for PCR based assays that use bisulfite converted DNA as input material, including bisulfite sequencing, methylation-specific PCR (MSP), combined bisulfite restriction analysis (COBRA), bisulfite-PCR-SCCP (BiPS), methylation-sensitive single-nucleotide primer extension (Ms-SNuPE), and PCR melting curve analysis. For a review of these methods, see Liu and Maekawa, Analytical Biochemistry, 2003, 317:259-265 (abstract).

methBLAST is based on NCBI's standalone WWW BLAST server, and searches custom databases generated by an in house developed perl script, which simulates CpG methylation and bisulfite modification. Because of this modification, the two daughter strands of any given sequence are no longer complementary after treatment. As either strand can serve as template for subsequent PCR amplification, the script performs the methylation and bisulfite modification on both strands (forward and reverse complement), and thus generates 4 derivative methBLAST databases for each original database, which are simultaneously queried to assess primer specificity:
  • bisulfite treated unmethylated sequences (unmethylated, forward) are annotated by [BISUL_UNMETH_FW], whereby all C's are changed to T's
  • bisulfite treated methylated forward sequence [BISUL_METH_FW] where all C's are changed to T's, except for those followed by a G (C's in CpG dinucleotides are protected by the methyl group)
  • bisulfite treated and unmethylated reverse complement sequence [BISUL_UNMETH_RC] where all C's in the reverse complement sequence are changed to T's
  • bisulfite treated and methylated reverse complement sequence [BISUL_METH_RC] where all C's in the reverse complement sequence are changed to T's, except for those followed by a G (C's in CpG dinucleotides are protected by the methyl group) 
    example:
agctagccagtcga   original sequence (forward, FW)
tcgactggctagct   reverse complement sequence (RC)
agttagttagttga   bisul_unmeth_FW
agttagttagtcga   bisul_meth_FW
ttgattggttagtt   bisul_unmeth_RC
tcgattggttagtt   bisul_meth_RC