Variants of a Thermus Aquaticus DNA Polymerase with Increased Selectivity for Applications in Allele- and Methylation-specific Amplification

Overview

Journal PLoS One

Specialties General Medicine
Science

Date 2014 May 8

PMID 24800860

Citations 12

Authors

Matthias Drum

Ramon Kranaster

Christina Ewald

Rainer Blasczyk

Andreas Marx

Affiliations

Soon will be listed here.

Abstract

The selectivity of DNA polymerases is crucial for many applications. For example, high discrimination between the extension of matched versus mismatched primer termini is desired for the detection of a single nucleotide variation at a particular locus within the genome. Here we describe the generation of thermostable mutants of the large fragment of Thermus aquaticus DNA polymerase (KlenTaq) with increased mismatch extension selectivity. In contrast to previously reported much less active KlenTaq mutants with mismatch discrimination abilities, many of the herein discovered mutants show conserved wild-type-like high activities. We demonstrate for one mutant containing the single amino acid exchange R660V the suitability for application in allele-specific amplifications directly from whole blood without prior sample purification. Also the suitability of the mutant for methylation specific amplification in the diagnostics of 5-methyl cytosines is demonstrated. Furthermore, the identified mutant supersedes other commercially available enzymes in human leukocyte antigen (HLA) analysis by sequence-specific primed polymerase chain reactions (PCRs).

Citing Articles

Enhancing the reverse transcriptase function in Taq polymerase via AI-driven multiparametric rational design.

Tomilova Y, Russkikh N, Yi I, Shaburova E, Tomilov V, Pyrinova G Front Bioeng Biotechnol. 2024; 12:1495267.

PMID: 39720166 PMC: 11666352. DOI: 10.3389/fbioe.2024.1495267.

Exploring the Impact of Primer-Template Mismatches on PCR Performance of DNA Polymerases Varying in Proofreading Activity.

Huang K, Zhang J, Li J, Qiu H, Wei L, Yang Y Genes (Basel). 2024; 15(2).

PMID: 38397205 PMC: 10888005. DOI: 10.3390/genes15020215.

Modified Taq DNA Polymerase for Allele-Specific Ultra-Sensitive Detection of Genetic Variants.

Lim Y, Park I, Lee H, Baek K, Lee B, Cho G J Mol Diagn. 2022; 24(11):1128-1142.

PMID: 36058471 PMC: 9746316. DOI: 10.1016/j.jmoldx.2022.08.002.

Inclusion of double helix structural oligonucleotide (STexS) results in an enhance of SNP specificity in PCR.

Kim J, Park H, Kyoung A, Park I, Lim S, Park B Sci Rep. 2021; 11(1):19098.

PMID: 34580382 PMC: 8476546. DOI: 10.1038/s41598-021-98610-8.

Rapid repair of human disease-specific single-nucleotide variants by One-SHOT genome editing.

Yokouchi Y, Suzuki S, Ohtsuki N, Yamamoto K, Noguchi S, Soejima Y Sci Rep. 2020; 10(1):13927.

PMID: 32811847 PMC: 7435196. DOI: 10.1038/s41598-020-70401-7.

References

Peltonen L, Altshuler D, de Bakker P, Deloukas P, Gabriel S, Gwilliam R . Integrating common and rare genetic variation in diverse human populations. Nature. 2010; 467(7311):52-8. PMC: 3173859. DOI: 10.1038/nature09298. View

Jorde L, Watkins W, Bamshad M, Dixon M, Ricker C, Seielstad M . The distribution of human genetic diversity: a comparison of mitochondrial, autosomal, and Y-chromosome data. Am J Hum Genet. 2000; 66(3):979-88. PMC: 1288178. DOI: 10.1086/302825. View

Novembre J, Johnson T, Bryc K, Kutalik Z, Boyko A, Auton A . Genes mirror geography within Europe. Nature. 2008; 456(7218):98-101. PMC: 2735096. DOI: 10.1038/nature07331. View

Sandoval J, Esteller M . Cancer epigenomics: beyond genomics. Curr Opin Genet Dev. 2012; 22(1):50-5. DOI: 10.1016/j.gde.2012.02.008. View

Franklin M, Wang J, Steitz T . Structure of the replicating complex of a pol alpha family DNA polymerase. Cell. 2001; 105(5):657-67. DOI: 10.1016/s0092-8674(01)00367-1. View

Strerath M, Cramer J, Restle T, Marx A . Implications of active site constraints on varied DNA polymerase selectivity. J Am Chem Soc. 2002; 124(38):11230-1. DOI: 10.1021/ja027060k. View

McKeown P, Merlini P, Berzuini C, Bernardinelli L, Peyvandi F, Tubaro M . Genome-wide association of early-onset myocardial infarction with single nucleotide polymorphisms and copy number variants. Nat Genet. 2009; 41(3):334-41. PMC: 2681011. DOI: 10.1038/ng.327. View

Bunce M, ONeill C, Barnardo M, Krausa P, Browning M, Morris P . Phototyping: comprehensive DNA typing for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 & DQB1 by PCR with 144 primer mixes utilizing sequence-specific primers (PCR-SSP). Tissue Antigens. 1995; 46(5):355-67. DOI: 10.1111/j.1399-0039.1995.tb03127.x. View

Dawson M, Kouzarides T . Cancer epigenetics: from mechanism to therapy. Cell. 2012; 150(1):12-27. DOI: 10.1016/j.cell.2012.06.013. View

10.

Abecasis G, Auton A, Brooks L, DePristo M, Durbin R, Handsaker R . An integrated map of genetic variation from 1,092 human genomes. Nature. 2012; 491(7422):56-65. PMC: 3498066. DOI: 10.1038/nature11632. View

11.

Kranaster R, Marx A . Taking fingerprints of DNA polymerases: multiplex enzyme profiling on DNA arrays. Angew Chem Int Ed Engl. 2009; 48(25):4625-8. DOI: 10.1002/anie.200900953. View

12.

Shively L, Chang L, LeBon J, Liu Q, Riggs A, Singer-Sam J . Real-time PCR assay for quantitative mismatch detection. Biotechniques. 2003; 34(3):498-502, 504. DOI: 10.2144/03343st01. View

13.

Papp A, Pinsonneault J, Cooke G, Sadee W . Single nucleotide polymorphism genotyping using allele-specific PCR and fluorescence melting curves. Biotechniques. 2003; 34(5):1068-72. DOI: 10.2144/03345dd03. View

14.

Wartiovaara A, Syvanen A . Analysis of nucleotide sequence variations by solid-phase minisequencing. Methods Mol Biol. 2002; 187:57-63. DOI: 10.1385/1-59259-273-2:057. View

15.

Strerath M, Marx A . Genotyping--from genomic DNA to genotype in a single tube. Angew Chem Int Ed Engl. 2005; 44(48):7842-9. DOI: 10.1002/anie.200501444. View

16.

Firth A, Patrick W . Statistics of protein library construction. Bioinformatics. 2005; 21(15):3314-5. DOI: 10.1093/bioinformatics/bti516. View

17.

Copeland W, Wang T . Mutational analysis of the human DNA polymerase alpha. The most conserved region in alpha-like DNA polymerases is involved in metal-specific catalysis. J Biol Chem. 1993; 268(15):11028-40. View

18.

Minnick D, Bebenek K, Osheroff W, Turner Jr R, Astatke M, Liu L . Side chains that influence fidelity at the polymerase active site of Escherichia coli DNA polymerase I (Klenow fragment). J Biol Chem. 1999; 274(5):3067-75. DOI: 10.1074/jbc.274.5.3067. View

19.

Relling M, Dervieux T . Pharmacogenetics and cancer therapy. Nat Rev Cancer. 2002; 1(2):99-108. DOI: 10.1038/35101056. View

20.

Guo Z, Liu Q, Smith L . Enhanced discrimination of single nucleotide polymorphisms by artificial mismatch hybridization. Nat Biotechnol. 1997; 15(4):331-5. DOI: 10.1038/nbt0497-331. View