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).

We describe the cloning and characterization of a mutated thermostable DNA polymerase from Thermus aquaticus (Taq) that exhibits an increased reverse transcriptase activity and is therefore designated for one-step PCR pathogen detection using established real-time detection methods. We demonstrate that this Taq polymerase mutant (Taq M1) has similar PCR sensitivity and nuclease activity as the respective Taq wild-type DNA polymerase. In addition, and in marked contrast to the wild-type, Taq M1 exhibits a significantly increased reverse transcriptase activity especially at high temperatures (>60 degrees C). RNA generally hosts highly stable secondary structure motifs, such as hairpins and G-quadruplexes, which complicate, or in the worst case obviate, reverse transcription (RT). Thus, RT at high temperatures is desired to weaken or melt secondary structure motifs. To demonstrate the ability of Taq M1 for RNA detection of pathogens, we performed TaqMan probe-based diagnostics of Dobrava viruses by one-step RT-PCR. We found similar detection sensitivities compared to commercially available RT-PCR systems without further optimization of reaction parameters, thus making this enzyme highly suitable for any PCR probe-based RNA detection method.

The epigenetic control of genes by the methylation of cytosine resulting in 5-methylcytosine (5mC) has fundamental implications for human development and disease. Analysis of alterations in DNA methylation patterns is an emerging tool for cancer diagnostics and prognostics. Here we report that two thermostable DNA polymerases, namely the DNA polymerase KlenTaq derived from Thermus aquaticus and the KOD DNA polymerase from Thermococcus kodakaraensis, are able to extend 3′-mismatched primer strands more efficiently from 5 mC than from unmethylated C. This feature was advanced by generating a DNA polymerase mutant with further improved 5mC/C discrimination properties and its successful application in a novel methylation-specific PCR approach directly from untreated human genomic DNA.

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Methylierung von Cytosin zu 5-Methylcytosin (5mC) dient der epigenetischen Kontrolle der Gentranskription in Säugetieren und hat direkte Auswirkungen auf die Entwicklung und Pathogenese des Menschen. Insbesondere die Diagnose und Prognostik von Krebspatienten profitierte in den letzten Jahren enorm von der Entwicklung neuer Methoden zur Analyse von DNA-Methylierungsmustern. Wir zeigen hier, dass zwei hitzestabile DNA-Polymerasen -die DNAPolymerase KlenTaq aus Thermus aquaticus und die KOD-DNA-Polymerase aus Thermococcus kodakaraensis -3'-fehlgepaarte Oligonukleotid-Primer gegenüber 5mC effektiver verlängern als gegenüber nichtmethyliertem C. Wir konnten außerdem eine DNA-Polymerase-Mutante mit weiter verbesserter 5mC/C-Diskriminierung erzeugen. Diese Mutante wurde erfolgreich in einer neuartigen 5mC-Detektierungsmethode eingesetzt, die es ermçglicht, den Methylierungszustand eines Cytosins direkt durch PCR von unbehandelter genomischer DNA zu bestimmen.