Fidelity of DNA polymerases in DNA amplification.

TLDR

Denaturing gradient gel electrophoresis was employed to separate PCR products, enabling direct enumeration and identification of point mutations introduced by T4, modified T7, Klenow fragment, and Taq polymerases and calculation of their average error rates. The error rates after 10⁶‑fold amplification were 3.4×10⁻⁵ for modified T7, 1.3×10⁻⁴ for Klenow fragment, 2.1×10⁻⁴ for Taq, and ≤3×10⁻⁶ for T4, with predominant transitions G·C→A·T for T4 and modified T7, A·T→G·C for Taq, and both transitions and 2–4 bp deletions for Klenow.

Abstract

Denaturing gradient gel electrophoresis (DGGE) was used to separate and isolate the products of DNA amplification by polymerase chain reaction (PCR). The strategy permitted direct enumeration and identification of point mutations created by T4, modified T7, Klenow fragment of polymerase I, and Thermus aquaticus (Taq) DNA polymerases. Incorrectly synthesized sequences were separated from the wild type by DGGE as mutant/wild-type heteroduplexes and the heteroduplex fraction was used to calculate the average error rate (mutations per base duplication). The error rate induced in the 104-base-pair low-temperature melting domain of exon 3 of the human hypoxanthine/guanine phosphoribosyltransferase (HPRT) gene was approximately 3.4 x 10(-5) for modified T7, 1.3 x 10(-4) for Klenow fragment, and 2.1 x 10(-4) for Taq polymerases after a 10(6)-fold amplification. The error rate for T4 DNA polymerase was not more than 3 x 10(-6) error per base duplication. The predominant mutations were sequenced and found to be transitions of G.C to A.T for T4 and modified T7 DNA polymerases, and A.T to G.C for Taq polymerase. Klenow fragment induced both possible transitions and deletions of 2 and 4 base pairs.