Predominant mutations induced by the Thermococcus litoralis, vent DNA polymerase during DNA amplification in vitro.

Abstract

We have analyzed the predominant mutations created during DNA amplification by PCR utilizing a DNA polymerase isolated from the Thermococcus litoralis (Vent DNA polymerase). Exon 3 of the human hypoxanthine guanine phosphoribosyl transferase (HPRT) gene was amplified using conditions optimized for efficiency of DNA amplification. The resulting PCR product was subjected to denaturing gradient gel electrophoresis (DGGE) to separate polymerase-induced mutant sequences from correctly amplified sequences. The nature of induced mutations was determined by isolating and sequencing the mutant sequences from the gel. Eighteen predominant mutations were found in the 104-bp low temperature melting domain of exon 3 and consisted of 16 A/T to G/C transitions, a G/C to T/A transversion and a complex 4-bp deletion. Thus, the Vent exonuclease proofreading activity seems to affect all misincorporation events with apparently equal probability (i.e., by a factor of five). The comparison of the error rates between analogues of Vent DNA polymerase proficient and deficient in the proofreading 3'-->5' exonuclease activity indicates that the lack of proofreading resulting in an approximate five-fold increase in induced error rate. However, the similarity of the patterns of the mutant sequences observed in DGGE suggested that both enzymes created predominantly the same kinds of mutations and at the same positions in this DNA template under the in vitro reaction conditions studied. This predominance of A/T to G/C transition is also a characteristic of the Taq DNA polymerase, although the positions of most errors induced by both enzymes are not identical.