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

CAACCCCAAACCCACAACCATAA

[15] | -

Methylated DNA

TTATTAGAGGGTGGGGCGGATCGC

GACCCCCGAACCGCGACCCTAA

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.

Pattyn et al. BMC Bioinformatics 2006 7:496   doi:10.1186/1471-2105-7-496