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Proc Natl Acad Sci U S A. 1989 Dec; 86(23): 9253–9257.
PMCID: PMC298472

Fidelity of DNA polymerases in DNA amplification.

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.

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  • Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. [PubMed]
  • Mullis KB, Faloona FA. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335–350. [PubMed]
  • Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. [PubMed]
  • Wrischnik LA, Higuchi RG, Stoneking M, Erlich HA, Arnheim N, Wilson AC. Length mutations in human mitochondrial DNA: direct sequencing of enzymatically amplified DNA. Nucleic Acids Res. 1987 Jan 26;15(2):529–542. [PMC free article] [PubMed]
  • McMahon G, Davis E, Wogan GN. Characterization of c-Ki-ras oncogene alleles by direct sequencing of enzymatically amplified DNA from carcinogen-induced tumors. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4974–4978. [PMC free article] [PubMed]
  • Wong C, Dowling CE, Saiki RK, Higuchi RG, Erlich HA, Kazazian HH., Jr Characterization of beta-thalassaemia mutations using direct genomic sequencing of amplified single copy DNA. Nature. 330(6146):384–386. [PubMed]
  • Muzyczka N, Poland RL, Bessman MJ. Studies on the biochemical basis of spontaneous mutation. I. A comparison of the deoxyribonucleic acid polymerases of mutator, antimutator, and wild type strains of bacteriophage T4. J Biol Chem. 1972 Nov 25;247(22):7116–7122. [PubMed]
  • Bessman MJ, Muzyczka N, Goodman MF, Schnaar RL. Studies on the biochemical basis of spontaneous mutation. II. The incorporation of a base and its analogue into DNA by wild-type, mutator and antimutator DNA polymerases. J Mol Biol. 1974 Sep 15;88(2):409–421. [PubMed]
  • Loeb LA, Kunkel TA. Fidelity of DNA synthesis. Annu Rev Biochem. 1982;51:429–457. [PubMed]
  • Kunkel TA, Loeb LA, Goodman MF. On the fidelity of DNA replication. The accuracy of T4 DNA polymerases in copying phi X174 DNA in vitro. J Biol Chem. 1984 Feb 10;259(3):1539–1545. [PubMed]
  • Scharf SJ, Horn GT, Erlich HA. Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science. 1986 Sep 5;233(4768):1076–1078. [PubMed]
  • Fischer SG, Lerman LS. DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: correspondence with melting theory. Proc Natl Acad Sci U S A. 1983 Mar;80(6):1579–1583. [PMC free article] [PubMed]
  • Lerman LS, Fischer SG, Hurley I, Silverstein K, Lumelsky N. Sequence-determined DNA separations. Annu Rev Biophys Bioeng. 1984;13:399–423. [PubMed]
  • Lerman LS, Silverstein K. Computational simulation of DNA melting and its application to denaturing gradient gel electrophoresis. Methods Enzymol. 1987;155:482–501. [PubMed]
  • Myers RM, Maniatis T, Lerman LS. Detection and localization of single base changes by denaturing gradient gel electrophoresis. Methods Enzymol. 1987;155:501–527. [PubMed]
  • Myers RM, Lumelsky N, Lerman LS, Maniatis T. Detection of single base substitutions in total genomic DNA. Nature. 1985 Feb 7;313(6002):495–498. [PubMed]
  • Noll WW, Collins M. Detection of human DNA polymorphisms with a simplified denaturing gradient gel electrophoresis technique. Proc Natl Acad Sci U S A. 1987 May;84(10):3339–3343. [PMC free article] [PubMed]
  • Cariello NF, Scott JK, Kat AG, Thilly WG, Keohavong P. Resolution of a missense mutant in human genomic DNA by denaturing gradient gel electrophoresis and direct sequencing using in vitro DNA amplification: HPRT Munich. Am J Hum Genet. 1988 May;42(5):726–734. [PMC free article] [PubMed]
  • Keohavong P, Kat AG, Cariello NF, Thilly WG. DNA amplification in vitro using T4 DNA polymerase. DNA. 1988 Jan-Feb;7(1):63–70. [PubMed]
  • Skopek TR, Liber HL, Penman BW, Thilly WG. Isolation of a human lymphoblastoid line heterozygous at the thymidine kinase locus: possibility for a rapid human cell mutation assay. Biochem Biophys Res Commun. 1978 Sep 29;84(2):411–416. [PubMed]
  • Keohavong P, Wang CC, Cha RS, Thilly WG. Enzymatic amplification and characterization of large DNA fragments from genomic DNA. Gene. 1988 Nov 15;71(1):211–216. [PubMed]
  • Tabor S, Richardson CC. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4767–4771. [PMC free article] [PubMed]
  • Tabor S, Huber HE, Richardson CC. Escherichia coli thioredoxin confers processivity on the DNA polymerase activity of the gene 5 protein of bacteriophage T7. J Biol Chem. 1987 Nov 25;262(33):16212–16223. [PubMed]
  • Patel PI, Nussbaum RL, gramson PE, Ledbetter DH, Caskey CT, Chinault AC. Organization of the HPRT gene and related sequences in the human genome. Somat Cell Mol Genet. 1984 Sep;10(5):483–493. [PubMed]
  • Tindall KR, Kunkel TA. Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase. Biochemistry. 1988 Aug 9;27(16):6008–6013. [PubMed]
  • Dunning AM, Talmud P, Humphries SE. Errors in the polymerase chain reaction. Nucleic Acids Res. 1988 Nov 11;16(21):10393–10393. [PMC free article] [PubMed]

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