The PCR plateau phase - towards an understanding of its limitations
The PCR plateau phase - towards an understanding of its limitations
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DOI：
10.1016/s0167-4781(00)00200-1
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发表时间：
2000-11-15
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影响因子：
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通讯作者：
Kainz, P
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文献类型：
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作者：
Kainz, P
最近发现来自水生栖热菌（Thermus aquaticus）和黄栖热菌（Thermus flavus）的DNA聚合酶可与短双链DNA结合。不具有序列特异性的片段[Kainz等人（2000）Biotechniques 28，278-82]。在本研究中，它表明，在后来的PCR循环扩增产物的积累也发挥了抑制作用的几种酶测试。为了模拟后面的循环条件，在存在不同量的1 kb双链DNA片段的情况下扩增来自噬菌体λ DNA的1.7kb序列。发现完全抑制Taq、Tfl和Pwo DNA聚合酶需要相对于聚合酶分子过量30倍摩尔的片段。当使用0.5、1或1.5 U/50穆尔反应体积的聚合酶浓度进行PCR扩增时，该化学计量关系保持恒定。完全抑制所需的1 kb DNA片段的量与对照（未添加片段）的产物产率相似，其运行至平台期水平。此外，通过使用发夹延伸测定，比较经历不同循环数的PCR混合物延伸3 '-凹陷末端的能力。发现在随后的PCR循环中积累的内源性扩增子DMA的存在完全抑制DNA聚合酶的活性。仍处于准线性相的PCR混合物部分地延伸了发夹。在这两种情况下，进一步添加聚合酶显着改善了它们的功能。这些结果表明，导致PCR平台期的主要因素是DNA聚合酶与其扩增产物的结合。(C)2000 Elsevier Science B. V.保留所有权利。
The DNA polymerases from Thermus aquaticus and Thermus flavus were recently found to bind to short double-stranded DNA. fragments without sequence specificity [Kainz et al. (2000) Biotechniques 28, 278-82]. In the present study, it is shown that the accumulation of amplification products during later PCR cycles also exerts an inhibitory effect on several enzymes tested. To simulate later cycle conditions, a 1.7 kb sequence from phage lambda DNA was amplified in the presence of various amounts of a 1 kb double-stranded DNA fragment. A 30-fold molar excess of fragments to polymerase molecules was found to be required for a complete inhibition of Taq, Tfl and Pwo DNA polymerase. This stoichiometric relation remained constant when PCR amplifications were performed using polymerase concentrations of 0.5, 1 or 1.5 U/50 mul reaction volume. The amount of 1 kb DNA fragments required for a complete inhibition was similar to the product yield of the controls (no fragment added), that were run to plateau phase levels. Additionally, PCR mixtures, that were subjected to different numbers of cycles, were compared in their ability to extend 3'-recessed ends by using a hairpin extension assay. The presence of endogenous amplicon DMA accumulated in later PCR cycles was found to inhibit completely the activity of DNA polymerase. PCR mixtures still in quasi-linear phase partially extended the hairpins. In both cases, a further addition of polymerase significantly improved their function. These results indicate that the main factor contributing to the plateau phase in PCR consists of binding of DNA polymerase to its amplification products. (C) 2000 Elsevier Science B.V. All rights reserved.