Direct DNA amplification from crude clinical samples using a PCR enhancer cocktail and novel mutants of Taq.
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 Taq DNA polymerases.
Zhang
Zhian
Z
DNA Polymerase Technology Inc., 1508 South Grand Blvd., St. Louis, MO 63104, USA. zhang@klentaq.com
Kermekchiev
Milko B
MB
Barnes
Wayne M
WM
D000925:Anticoagulants / Q000378:metabolism; D004247:DNA / Q000032:analysis / Q000097:blood; D006493:Heparin / Q000378:metabolism; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000235:genetics / Q000378:metabolism
D000925:Anticoagulants; D006493:Heparin; D004247:DNA; D019914:Taq Polymerase