耐热Taq DNA聚合酶的酸酐修饰及其在降低PCR非特异性扩增中的应用评价

酸酐能与聚合酶上的赖氨酸结合形成聚合物,试验通过选用合适的酸酐,修饰Taq DNA聚合酶。在常温下,酶活性被封闭,升温过程中不表现酶活,从而减少非特异性扩增和引物二聚体的形成；而在较高的温度下一段时间后,一般为94℃、15 min,酸酐脱落,聚合酶得以恢复正常的扩增活性,从而实现PCR反应的热启动。由此建立的热启动PCR体系灵敏度高,可以检测到10个拷贝的DNA分子,较普通PCR体系提高了2个数量级。此方法较其他方法操作简单成本低廉,且所得的热启动Taq DNA聚合酶稳定性好,便于保存,其灵敏性与特异性也更高。这种通过化学修饰形成的复合物达到热启动PCR目的的方法的建立,具有着重要的意义,是提高PCR灵敏度和特异性的重要方法之一。

Chemical modification of heat-Taq DNA polymerase and evaluation of its effect on minimizing non-specific amplifications in PCR

Anhydride is capable of binding to and forming lysine polymer with DNA polymerase.Upon such modification, catalytic activity of Taq DNA polymerase is completely abolished during the preheating stage of PCR, thus minimizing non-specific amplifications and the formation of primer dimmers which interfere with efficient PCR amplification. Once exposed at high temperature for a while, such as 94℃ for 15min, anhydride could be removed from polymerase, resulting in almost full recovery of its catalytic activity, thus achieving hot-start PCR. Here in this paper, we chose an appropriate sort of anhydride to modify Taq DNA polymerase. We found that using such modified polymerase, sensitivity of PCR could be greatly improved by two orders of magnitude and 10 copies of the DNA molecule is now achievable for detection. In addition, the course of such modification was easy to handle and less cost. Moreover, modified polymerase was more stable, easy to store and show higher sensitivity and specificity. Therefore, the establishment of hot-start PCR system through chemical modification became one of the most crucial approaches to elevate the sensitivity and specificity for PCR, and was of great application value in the near future．