Real time PCR of prothrombin factor II Human gDNA with KOD pol wildtype and mutants.

Real time PCR curves when using a forward primer that terminates opposite the SNP site in either a matched or mismatched fashion, depending on the template. (A) real time PCR curves obtained when using KOD pol wildtype. No discrimination is seen for either of the DNA samples: G-wt (genotype G/G), A-heterozygous (genotype G/A) or A-homozygous (genotype A/A). (B)+(C)+(D) real time PCR curves when using KOD pols R501C, R606Q, and R606W, respectively. Discrimination is seen for the amplification of the A-homozygous (A/A) Factor II template which is severely slowed down when using the KOD mutants. (E) agarose gel electrophoresis analyzing PCR products from (B)-(D). M = marker. PCR products are only visible in samples containing template DNA with at least one allele that results in a matched primer-template duplex (C-primer with G/G and G/A DNA template); the homozygous A/A template does not yield a PCR product.