Extension of base mispairs by Taq DNA polymerase: implications for single nucleotide discrimination in PCR
Open accessAbstract
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.
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Primer-extension following a mismatch can be much slower than following a Watson-Crick base-pair, and this post-mismatch stalling is significant in non-enzymatic DNA template-copying model systems.
“Primer-extension following a mismatch can be much slower than following a Watson-Crick base-pair. Such post-mismatch stalling was originally noticed in enzymatic reactions [31,32] but is also significant in a non-enzymatic DNA template-copying model system [33].”
From The eightfold path to non-enzymatic RNA replication (2012) · cites this paper as [32]
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- Authors
- Huang, M.-M., Arnheim, N., & Goodman, M. F.
- Affiliations
- University of Southern California (US)
- Corresponding author
- Goodman, Myron F.
- Venue
- Nucleic Acids Research
- Publisher
- Oxford University Press (OUP)
- Published
- 1992
- Volume
- 20
- Issue
- 17
- Pages
- 4567-4573
- ISSN
- 0305-1048 (ISSN-L)
- 1362-4962
- PMC
- PMC334186
- OpenAlex
- W2072194704
- Cited by
- 365
- References
- 28
- Citations by year
- 2026: 6 · 2025: 4 · 2024: 6 · 2023: 16 · 2022: 9 · 2021: 4 · 2020: 6 · 2019: 4 · 2018: 8 · 2017: 7 · 2016: 11 · 2015: 12 · 2014: 10 · 2013: 14 · 2012: 17
- FWCI
- 3.57
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Nucleic Acids Research (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institutes of Health (5r01gm042554-13, GM21422)
- National Institute of General Medical Sciences (GM 42554, GM-36745, GM 21422)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Keywords
- Taq DNA polymerase, mismatch extension, single-nucleotide discrimination, PCR, enzyme affinity, Thermus aquaticus
- MeSH terms
- Base Composition, Base Sequence, DNA-Directed DNA Polymerase, Molecular Sequence Data, Nucleotides, Oligodeoxyribonucleotides, Temperature, Polymerase Chain Reaction, Taq Polymerase
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