title: Variants of a Thermus aquaticus DNA polymerase with increased selectivity for applications in allele- and methylation-specific amplification.
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).
authors.affiliation: Department of Chemistry, Konstanz Research School Chemical Biology, University of Konstanz, Konstanz, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Drum
authors.affiliation: Department of Chemistry, Konstanz Research School Chemical Biology, University of Konstanz, Konstanz, Germany; myPOLS Biotec, University of Konstanz, Konstanz, Germany.
authors.forename: Ramon
authors.initials: R
authors.lastname: Kranaster
authors.affiliation: Department of Chemistry, Konstanz Research School Chemical Biology, University of Konstanz, Konstanz, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Ewald
authors.affiliation: Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Blasczyk
authors.affiliation: Department of Chemistry, Konstanz Research School Chemical Biology, University of Konstanz, Konstanz, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Marx
mesh_terms: D000483:Alleles; D019943:Amino Acid Substitution; D020137:Base Pair Mismatch; D019175:DNA Methylation; D015894:Genome, Human; D006680:HLA Antigens / Q000032:analysis; D006367:HeLa Cells; D006650:Histocompatibility Testing; D006801:Humans; D007700:Kinetics; D020641:Polymorphism, Single Nucleotide; D017434:Protein Structure, Tertiary; D011516:Prothrombin / Q000235:genetics; D019914:Taq Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D006680:HLA Antigens; D011516:Prothrombin; D019914:Taq Polymerase