Real time PCR of a synthetic prothrombin Factor II oligonucleotide with KOD wildtype and mutants.
(A) primer and (partial) template sequence from the prothrombin Factor II gene. The 3’ end of the primer was either matched or mismatched to the template. (B) real time PCR curves obtained when using KOD pol wildtype. The enzyme distinguishes only little when amplifying from matched (C-G) or mismatched (C-A) primer-template duplex. (C) + (D) + (E) real time PCR curves obtained by using KOD R501C, KOD R606Q and KOD R606W respectively. Increased discrimination is shown, as indicated by slower PCR amplification from mismatched (C-A) primer-template duplexes. R501C barely reaches saturation after 30 cycles; both R606 variants are almost inactive in the mismatch case under the applied conditions.
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