The new technology of hot start polymerase chain reaction

Abstract

The use of monoclonal antibodies against Taq DNA polymerase in the polymerase chain reaction is proposed. These antibodies effectively inhibit polymerase activity at temperatures <70~ activity being restored upon DNA melting in the very first stage of amplification. As in the "hot start" reaction, the addition of the antibodies to the incubation medium markedly improves the reaction yield. The method is particularly effective for the identification of a few copies of DNA.

Analysis of mitochondrial DNA heteroplasmy of fertile and male-sterile sugar beet plants (Beta vulgaris)

“…Amplification of the normalization coxII and rps4 gene markers was optimized by adjusting primer and magnesium concentrations in the reaction mixture, as well as by using a hot start based on the addition of monoclonal antibodies to the reaction mixture, which blocks Taq polymerase activity (Rybalkin et al, 1996). The estimation of the dynamic diapason showed that under the optimized conditions the standard curve remains linear (R 2 > 0.99) in the range from 100 to 10 8 copies per reaction and the sensitivity of the detection is 50 copies of a marker sequence per 1 g of plant tis sue, which considerably enhanced the sensitivity of the methods that were previously used for the estima tion of sugar beet mtDNA heteroplasmy (Bragin et al, 2006;Cheng et al, 2009).…”

Unexpected Mechanism and Inhibition Effect for Nonspecific Amplification Involving Dynamic Binding of Primers with Background DNA

“…Nonspecific DNA amplification produces unexpected replicons from off-target sequences, usually causing severe issues for highly sensitive and specific target detection, − especially when millions and even billions of background DNA (bgDNA) molecules are present. , It commonly occurs in various amplification methods, including polymerase chain reaction (PCR), , loop-mediated isothermal amplification (LAMP), , and rolling circle amplification (RCA) . To avoid nonspecific amplification, the mechanism has to be clarified so that we can focus on the key points of the development of suppression methods. − However, although several challenges have been carried out, no satisfactory result has been obtained. , Usually, researchers attribute nonspecific amplification to the overlap-extension of primers, i.e., the short complementary parts at 3′-ends between two primers hybridize and extend by DNA polymerase. − Obviously, in this case, the nonspecific products should be shorter than the sum of the two primers (<40 bp). Contradictorily, most of the so-called primer dimers (nonspecific products in PCR) are longer, ranging from 50 to 150 bp. − In addition, this mechanism cannot explain that nonspecific amplification is hard to avoid even when primers are well designed …”