Fig 1.
(A) Crystal structure of a binary complex of KOD pol (PDB code 4K8Z [48]. The sections shown in detail, illustrate how the investigated amino acids interact with the primer or template backbone. (B) Schematic drawing of the primer-template duplex. Amino acids and their respective interactions with the DNA substrate are indicated by arrows according to [48].
Fig 2.
Results of screening the KOD pol library as activity chart.
Amino acids of interest are given on the left, amino acid exchanges are depicted at the top in single letter code. Colors indicate activity of mutant proteins measured as Ct values in real time PCR screening assays using bacterial lysates. Darker shades of blue indicate lower Ct values, grey shows PCR inactive mutants. Boxes marked with asterisks show variants that could not be obtained during several rounds of cloning.
Fig 3.
Single nucleotide primer extension experiments using KOD pol wildtype and mutants.
(A) primer and (partial) template sequence from the prothrombin factor II gene. The 3’ end of the radioactively labeled primer was either matched or mismatched to the template. (B) PAGE analysis of single nucleotide extension of the primer by either KOD pol wildtype or mutants, as indicated. Reactions were carried out for the indicated times. (C) + (D) percentage of primer conversion vs time for match and mismatch case, respectively. Gel bands were quantified with the Quantity One software from BioRad, results are shown as means +/- SD of three independent experiments. Graphs were prepared with Origin2015.
Table 1.
Analysis of steady-state kinetics of dGMP incorporation after either matched or mismatched primer-template substrate.
Fig 4.
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.
Fig 5.
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.
Fig 6.
Real time PCR for cytosine methylation detection.
(A) The chosen forward primer terminates opposite either C or 5mC, depending on the template. Two forward primers were used, carrying either G or A at their 3’ end, resulting in matched or mismatched primer-template duplex respectively. (B) real time PCR curves using KOD pol wildtype. Equal amplification is observed for all four primer/template samples. (C) real time PCR curves using KOD pol R501C. Amplification from the matched primer is not distinguishable between C or 5mC. Amplification from the mismatched primer is in both cases slightly slower, but not significantly different between C and 5mC. (D) + (E) real time PCR curves using KOD pol R606Q and KOD pol R606W, respectively. Matched primer-template duplexes are amplified without discrimination and comparable to KOD pol wildtype. In the mismatch case, methylated DNA is clearly favored, demonstrating the ability of the enzyme variants to discriminate between C and 5mC.