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).
Highlights
Personalized medicine providing therapies, adapted to each patient’s genetic predisposition[1][2][3][4][5], is mainly supported by the analysis of single nucleotide polymorphisms (SNPs)[1]
This created a basis for the first human haplotype map (HapMap) project with more than one million SNPs for which accurate and complete genotypes have been obtained in 269 DNA samples from four populations[19]
Different techniques can be used for the analysis of SNPs such as selective primer extension, e.g. minisequencing[23][24][25], pyrosequencing[26] or allele specific amplification (ASA)[27][23][28]
Summary
Personalized medicine providing therapies, adapted to each patient’s genetic predisposition[1][2][3][4][5], is mainly supported by the analysis of single nucleotide polymorphisms (SNPs)[1]. Intergenic SNPs present interesting markers for the determination of parentage[11][12], anthropology[13][14] or forensic tasks e.g. genetic fingerprinting Many of these variations can affect predispositions for diseases or responses to drugs, chemicals and vaccines[1][15], which makes them especially interesting for pharmacogenomics[16][17]. ASA, unlike most other methods for SNP detection, does not require preliminary amplification of the target genetic material[29][30]. It combines amplification and detection in a single reaction, based on the discrimination of matched and mismatched primer/template complexes. In SNP diagnostics this provides information about the absence or presence of an SNP
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