• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of narLink to Publisher's site
Nucleic Acids Res. Sep 11, 1992; 20(17): 4567–4573.
PMCID: PMC334186

Extension of base mispairs by Taq DNA polymerase: implications for single nucleotide discrimination in PCR.

Abstract

Thermus aquaticus (Taq) DNA polymerase was used to measure the extension efficiency for all configurations of matched and mismatched base pairs at template-primer 3'-termini. The transition mispairs, A(primer).C, C.A, G.T, and T.G were extended 10(-3) to 10(-4)-fold less efficiently than their correctly paired counterparts. Relative efficiencies for extending transversion mispairs were 10(-4) to 10(-5) for T.C and T.T, about 10(-6) for A.A, and less than 10(-6) for G.A, A.G, G.G and C.C. The transversion mispair C(primer).T was extended with high efficiency, about 10(-2) compared to a correct A.T basepair. The unexpected ease of extending the C.T mismatch was not likely to have been caused by primer-template misalignment. Taq polymerase was observed to bind with similar affinities to each of the correctly paired and mispaired primer-template 3'-ends. Thus, the failure of Taq polymerase to extend mismatches efficiently appears to be an intrinsic property of the enzyme and not due to an inability to bind to 3'-terminal mispairs. For almost all of the mispairs, C.T being the exception, Taq polymerase exhibits about 100 to 1000-fold greater discrimination against mismatch extension compared to avian myeloblastosis reverse transcriptase and HIV-1 reverse transcriptase which extend most mismatched basepairs permissively. Relative mismatch extension efficiencies for Taq polymerase were measured at 45 degrees C, 55 degrees C and 70 degrees C and found to be independent of temperature. The mispair extension data should be important in designing experiments using PCR to distinguish between sequences that vary by a single nucleotide.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.6M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Arnheim N, Erlich H. Polymerase chain reaction strategy. Annu Rev Biochem. 1992;61:131–156. [PubMed]
  • Erlich HA, Gelfand D, Sninsky JJ. Recent advances in the polymerase chain reaction. Science. 1991 Jun 21;252(5013):1643–1651. [PubMed]
  • Petruska J, Goodman MF, Boosalis MS, Sowers LC, Cheong C, Tinoco I., Jr Comparison between DNA melting thermodynamics and DNA polymerase fidelity. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6252–6256. [PMC free article] [PubMed]
  • Perrino FW, Loeb LA. Differential extension of 3' mispairs is a major contribution to the high fidelity of calf thymus DNA polymerase-alpha. J Biol Chem. 1989 Feb 15;264(5):2898–2905. [PubMed]
  • Mendelman LV, Petruska J, Goodman MF. Base mispair extension kinetics. Comparison of DNA polymerase alpha and reverse transcriptase. J Biol Chem. 1990 Feb 5;265(4):2338–2346. [PubMed]
  • Creighton S, Huang MM, Cai H, Arnheim N, Goodman MF. Base mispair extension kinetics. Binding of avian myeloblastosis reverse transcriptase to matched and mismatched base pair termini. J Biol Chem. 1992 Feb 5;267(4):2633–2639. [PubMed]
  • Kunkel TA, Alexander PS. The base substitution fidelity of eucaryotic DNA polymerases. Mispairing frequencies, site preferences, insertion preferences, and base substitution by dislocation. J Biol Chem. 1986 Jan 5;261(1):160–166. [PubMed]
  • Kuchta RD, Benkovic P, Benkovic SJ. Kinetic mechanism whereby DNA polymerase I (Klenow) replicates DNA with high fidelity. Biochemistry. 1988 Sep 6;27(18):6716–6725. [PubMed]
  • Yu H, Goodman MF. Comparison of HIV-1 and avian myeloblastosis virus reverse transcriptase fidelity on RNA and DNA templates. J Biol Chem. 1992 May 25;267(15):10888–10896. [PubMed]
  • Longley MJ, Bennett SE, Mosbaugh DW. Characterization of the 5' to 3' exonuclease associated with Thermus aquaticus DNA polymerase. Nucleic Acids Res. 1990 Dec 25;18(24):7317–7322. [PMC free article] [PubMed]
  • Fisher PA, Korn D. Ordered sequential mechanism of substrate recognition and binding by KB cell DNA polymerase alpha. Biochemistry. 1981 Aug 4;20(16):4560–4569. [PubMed]
  • Detera SD, Becerra SP, Swack JA, Wilson SH. Studies on the mechanism of DNA polymerase alpha. Nascent chain elongation, steady state kinetics, and the initiation phase of DNA synthesis. J Biol Chem. 1981 Jul 10;256(13):6933–6943. [PubMed]
  • Tindall KR, Kunkel TA. Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase. Biochemistry. 1988 Aug 9;27(16):6008–6013. [PubMed]
  • Wong I, Patel SS, Johnson KA. An induced-fit kinetic mechanism for DNA replication fidelity: direct measurement by single-turnover kinetics. Biochemistry. 1991 Jan 15;30(2):526–537. [PubMed]
  • Kwok S, Kellogg DE, McKinney N, Spasic D, Goda L, Levenson C, Sninsky JJ. Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nucleic Acids Res. 1990 Feb 25;18(4):999–1005. [PMC free article] [PubMed]
  • Mendelman LV, Boosalis MS, Petruska J, Goodman MF. Nearest neighbor influences on DNA polymerase insertion fidelity. J Biol Chem. 1989 Aug 25;264(24):14415–14423. [PubMed]
  • Sowers LC, Fazakerley GV, Kim H, Dalton L, Goodman MF. Variation of nonexchangeable proton resonance chemical shifts as a probe of aberrant base pair formation in DNA. Biochemistry. 1986 Jul 15;25(14):3983–3988. [PubMed]
  • Boulard Y, Cognet JA, Gabarro-Arpa J, Le Bret M, Sowers LC, Fazakerley GV. The pH dependent configurations of the C.A mispair in DNA. Nucleic Acids Res. 1992 Apr 25;20(8):1933–1941. [PMC free article] [PubMed]
  • 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]
  • Wu DY, Ugozzoli L, Pal BK, Wallace RB. Allele-specific enzymatic amplification of beta-globin genomic DNA for diagnosis of sickle cell anemia. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2757–2760. [PMC free article] [PubMed]
  • Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith JC, Markham AF. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res. 1989 Apr 11;17(7):2503–2516. [PMC free article] [PubMed]
  • Gibbs RA, Nguyen PN, Caskey CT. Detection of single DNA base differences by competitive oligonucleotide priming. Nucleic Acids Res. 1989 Apr 11;17(7):2437–2448. [PMC free article] [PubMed]
  • Ehlen T, Dubeau L. Detection of ras point mutations by polymerase chain reaction using mutation-specific, inosine-containing oligonucleotide primers. Biochem Biophys Res Commun. 1989 Apr 28;160(2):441–447. [PubMed]
  • Li H, Cui X, Arnheim N. Direct electrophoretic detection of the allelic state of single DNA molecules in human sperm by using the polymerase chain reaction. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4580–4584. [PMC free article] [PubMed]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...