Magnesium precipitate hot start method for PCR
Magnesium precipitate hot start method for PCR
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DOI：
10.1006/mcpr.2002.0407
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发表时间：
2002-06-01
影响因子：
3.3
通讯作者：
Rowlyk, KR
中科院分区：
文献类型：
--
作者：
Barnes, WM;Rowlyk, KR
针对HIV-1 Gag基因的扩增和其他具有挑战性的靶基因的扩增，提出了一种简单的、完全由缓冲系统组成的新的热启动PCR方法。这种用于PCR的新型缓冲组合物和反应组装方案除了标准缓冲试剂外，还包括高浓度结合的镁和磷酸盐。由此产生的含镁沉淀物为PCR提供了一个热开始，因为沉淀物中的镁在热循环之前不能被DNA聚合酶获得。不需要在热循环仪上进行额外的操作或更改标准的热循环曲线。在正常循环中，镁在前3个循环中完全可用。该方法有效地防止了几种DNA聚合酶或被测试的DNA聚合酶的混合物，包括Klentaq1，KlentaqLA，PFU和全长野生型DNA聚合酶。一旦形成沉淀物，热启动缓冲器在-20度、4度或25度下稳定并至少工作一周。我们证明，对于需要热启动的几个靶基因扩增，该方法与手动热启动(在或高于引物的退火温度添加镁)一样有效。(C)20.02爱思唯尔科学有限公司。保留所有权利。
For the amplification of HIV-1 gag gene, and other challenging targets, a simple new hot start PCR protocol is presented which consists simply and entirely of the buffer system. This novel buffer composition and reaction assembly protocol for PCR includes magnesium and phosphate combined at high concentration in addition to standard buffer reagents. The resulting magnesium-containing precipitate provides a hot start for PCR, since the magnesium in the precipitate is unavailable to DNA polymerase until thermal cycling. No extra manipulations at the thermal cycler or changes to standard thermal cycling profiles are necessary. Upon normal cycling, the magnesium becomes fully available within the first 3 cycles. The method effectively prevents premature primer extension by several DNA polymerases or mixtures of DNA polymerases tested, including Klentaq1, KlentaqLA, Pfu, and full-length wild-type DNA polymerase. Once the precipitate is formed, the hot start buffer is stable and functional for at least a week at -20degrees, 4degrees, or 25degrees. We demonstrate that the method is as effective as a manual hot start (addition of magnesium at or above primers' annealing temperatures) for several target gene amplifications which require a hot start. (C) 20.02 Elsevier Science Ltd. All rights reserved.