Figure 4.

The mutational spectrum of Qβ replicase, Mut II, and EP-PCR. (A): Qβ replicase did not have significant bias for A/T->N/N (black bars) or G/C->N/N (grey bars) changes. Mut II and EP-PCR using either Mn²⁺ (manufacture's protocol 3) or Mn²⁺ with an unbalanced dGTP concentration (manufacture's protocol 7) demonstrated significant A/T->N/N bias. (B): The different transition/transversion ratios between the methods. Qβ replicase and Mut II did not show a significant bias for transitions (black bars) over transversions (grey bars) compared to both versions of EP-PCR, which showed a significant preference for transitions. (C): The percentage of all possible base substitutions for each of the mutagenesis methods analyzed. All possible base substitutions were recovered with Qβ replicase. The G/C -> C/G transversion was only recovered once with Mut II, which also over represented A/T-> T/A transversions. With EP-PCR (manufacture's protocol 3) A/T-> T/A transversions were over represented, the A/T->C/G transversion was only recovered once and the G/C -> C/G transversion was not recovered. G/C -> C/G and G/C->T/A transversions were not recovered with EP-PCR (manufacture's protocol 7). Both versions of EP-PCR over represented A/T->G/C transitions. Experiments were repeated 2 times with the standard deviation between experiments shown as error bars. The data set used to generate Figure 3 was as follows: 71 point mutations were characterized for Qβ replicase (52,327 bases were sequenced in total with an average mutation rate of 1 point mutation every 737 bases giving a mutation frequency of 1.35). 48 point mutations were characterized for Mut II (6720 bases were sequenced in total with a mutation rate of 1/140 bases giving a mutation frequency of 7.14). EP-PCR manufacture's protocol 3; 74 point mutations were characterized (36,960 bases were sequenced in total with a mutation rate of 1/499 bases and a mutation frequency of 2.00) and manufacture's protocol 7; 43 point mutations were characterized (9,600 bases were sequenced in total with a mutation rate of 1/225 bases giving a mutation frequency of 4.48).