Kellogg D E, Rybalkin I, Chen S, Mukhamedova N, Vlasik T, Siebert P D, Chenchik A
CLONTECH Laboratories, Palo Alto, CA.
Biotechniques. 1994 Jun;16(6):1134-7.
The specificity and DNA yield of PCRs are often improved by the "hot start" technique and analogous methods. The intent of the approach is to eliminate or prevent the generation of nonspecific PCR templates that may be synthesized at ambient temperature prior to thermal cycling. Monoclonal antibodies (MAbs) raised in mice to the purified DNA polymerase of Thermus aquaticus (Taq) were selected for their ability to reversibly block polymerase activity. The MAbs, incubated with Taq DNA polymerase and added to PCR tubes at ambient temperature, yield specific DNA fragments upon amplification when using high numbers of temperature cycles and a very low copy number of target DNA in a complex DNA background. This approach, using the TaqStart Antibody, permits the preparation of reaction mixtures at ambient temperatures without the subsequent opening of reaction tubes, use of grease or waxes, or of degradative enzymes and deoxyribonucleotide analogs.
聚合酶链反应(PCR)的特异性和DNA产量通常通过“热启动”技术及类似方法得以提高。该方法的目的是消除或防止在热循环之前于室温下可能合成的非特异性PCR模板的产生。针对水生栖热菌(Taq)纯化的DNA聚合酶在小鼠体内产生的单克隆抗体(MAbs),因其能够可逆地阻断聚合酶活性而被选用。这些单克隆抗体与Taq DNA聚合酶一起孵育,并在室温下添加到PCR管中,当在复杂DNA背景中使用大量温度循环和极低拷贝数的靶DNA进行扩增时,可产生特异性DNA片段。这种使用TaqStart抗体的方法允许在室温下制备反应混合物,而无需随后打开反应管、使用油脂或蜡,也无需使用降解酶和脱氧核苷酸类似物。