Paul Natasha, Shum Jonathan, Le Tony
Department of Research and Development, TriLink BioTechnologies, Inc., San Diego, CA, USA.
Methods Mol Biol. 2010;630:301-18. doi: 10.1007/978-1-60761-629-0_19.
Hot Start activation approaches are increasingly being used to improve the performance of PCR. Since the inception of Hot Start as a means of blocking DNA polymerase extension at lower temperatures, a number of approaches have been developed that target the essential reaction components such as magnesium ion, DNA polymerase, oligonucleotide primers, and dNTPs. Herein, five different Hot Start activation protocols are presented. The first method presents the use of barriers as a means of segregating key reaction components until a Hot Start activation step. The second and third protocols demonstrate Hot Start approaches to block DNA polymerase activity through the use of anti-DNA polymerase antibodies and accessory proteins, respectively. The fourth and fifth protocols utilize thermolabile chemical modifications to the oligonucleotide primers and dNTPs. The results presented demonstrate that all protocols significantly improve the specificity of traditional thermal cycling protocols.
热启动激活方法越来越多地用于提高聚合酶链反应（PCR）的性能。自从热启动作为一种在较低温度下阻断DNA聚合酶延伸的方法问世以来，已经开发了许多针对镁离子、DNA聚合酶、寡核苷酸引物和脱氧核苷三磷酸（dNTPs）等关键反应成分的方法。本文介绍了五种不同的热启动激活方案。第一种方法介绍了使用屏障将关键反应成分分离，直到进行热启动激活步骤。第二和第三种方案分别展示了通过使用抗DNA聚合酶抗体和辅助蛋白来阻断DNA聚合酶活性的热启动方法。第四和第五种方案利用对寡核苷酸引物和dNTPs的热不稳定化学修饰。给出的结果表明，所有方案都显著提高了传统热循环方案的特异性。
