methBLAST and methPrimerDB: web-tools for PCR based methylation analysis

BMC Bioinformatics

Table 2 Sequence comparison of primers from two CDKN2A MSP assays

Ref.\| methPrimerDB ID	Template	Forward primer sequence	Reverse primer sequence
[11] \| methPrimerDB ID 17	Unmethylated DNA	TTATTAGAGGGTGGGGTGGATTGT	CAACCCCAAACCACAACCATAA
[11] \| methPrimerDB ID 17	Methylated DNA	TTATTAGAGGGTGGGGCGGATCGC	GACCCCGAACCGCGACCGTAA
[15] \| -	Unmethylated DNA	TTATTAGAGGGTGGGGTGGATTGT	CAACCCCAAACCC ACAACCATAA
[15] \| -	Methylated DNA	TTATTAGAGGGTGGGGCGGATCGC	GACCCCC GAACCGCGACCC TAA

The primer pairs from methPrimerDB ID 17 can generate a specific PCR product and show correct alignment with the target sequence when performing a methBLAST analysis (see Table 1). The primers published in [15] have identical forward primer sequences but the reverse primer sequence for detecting unmethylated DNA contains a cytosine insert between positions 12 and 13 and the reverse primer sequence for detecting methylated DNA contains a cytosine insert between positions 6 and 7 and a substitution from guanine to cytosine at position 18 (see nucleotides in bold and underlined). These sequence errors make the assay non-functional.

ISSN: 1471-2105