Potent PCR inhibitors in blood and soil samples can cause false negative results from PCR-based clinical and forensic tests. We show that the effect of these inhibitors is primarily upon Taq DNA polymerase, since mutational alteration of the polymerase can overcome the inhibition to the extent that no DNA purification is now required. An N-terminal deletion (Klentaq1) is some 10–100-fold inhibition resistant to whole blood compared to full-length, wild-type (w.t.) Taq, which is strongly inhibited by 0.1–1% blood. Further mutations at codon 708, both in Klentaq 1 and Taq, confer enhanced resistance to various inhibitors of PCR reactions, including whole blood, plasma, hemoglobin, lactoferrin, serum IgG, soil extracts and humic acid, as well as high concentrations of intercalating dyes. Blood PCR inhibitors can predominantly reduce the DNA extension speed of the w.t. Taq polymerase as compared to the mutant enzymes. Single-copy human genomic targets are readily amplified from whole blood or crude soil extract, without pretreatment to purify the template DNA, and the allowed increase in dye concentration overcomes fluorescence background and quenching in real-time PCR of blood.
PCR-based clinical and forensic tests often have low sensitivity or even false-negative results caused by potent PCR inhibitors found in blood and soil. It is widely accepted that purification of target DNA before PCR is necessary for successful amplification. In an attempt to overcome PCR inhibition , enhance PCR amplification , and simplify the PCR protocol, we demonstrate improved PCR-enhancing cocktails containing nonionic detergent , L-carnitine , D-(؉)-trehalose , and heparin. These cocktails , in combination with two inhibitor-resistant Taq mutants , OmniTaq and Omni Klentaq , enabled efficient amplification of exogenous , endogenous , and high-GC content DNA targets directly from crude samples containing human plasma , serum , and whole blood without DNA purification. In the presence of these enhancer cocktails , the mutant enzymes were able to tolerate at least 25% plasma , serum , or whole blood and as high as 80% GC content templates in PCR reactions. These enhancer cocktails also improved the performance of the novel Taq mutants in real-time PCR amplification using crude samples , both in SYBR Green fluorescence detection and TaqMan assays. The novel enhancer mixes also facilitated DNA amplification from crude samples with various commercial
Although the thermophilic bacterium Thermus aquaticus grows optimally at 70 degrees C and cannot grow at moderate temperatures, its DNA polymerase I has significant activity at 20-37 degrees C. This activity is a bane to some PCRs, since it catalyzes non-specific priming. We report mutations of Klentaq (an N-terminal deletion variant) DNA polymerase that have markedly reduced activity at 37 degrees C yet retain apparently normal activity at 68 degrees C and resistance at 95 degrees C. The first four of these mutations are clustered on the outside surface of the enzyme, nowhere near the active site, but at the hinge point of a domain that has been proposed to move at each cycle of nucleotide incorporation. We show that the novel cold-sensitive mutants can provide a hot start for PCR and exhibit slightly improved fidelity.
The 5'-terminal coding sequence for the 37 S precursor to rRNA of Saccharomyces cerevisiae is identified by reverse transcriptase extension and protection mapping with nuclease S1. The sequence of a 419 bp rDNA fragment containing the transcription initiation site and its adjacent region is determined.
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