Discrimination of primer 3′-nucleotide mismatch by Taq DNA polymerase during polymerase chain reaction
Discrimination of primer 3′-nucleotide mismatch by Taq DNA polymerase during polymerase chain reaction
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DOI：
10.1006/abio.2000.4635
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发表时间：
2000-08-15
影响因子：
2.9
通讯作者：
Reis, RJS
中科院分区：
文献类型：
--
作者：
Ayyadevara, S;Thaden, JJ;Reis, RJS
我们考察了引物-模板不匹配对聚合酶链式反应效率的影响。对于以T、C或G为3‘核苷酸的引物，水热菌(Thermus Aquaticus，Taq)DNA聚合酶对该碱基具有高度的模板互补性，但相对于倒数第二个核苷酸的限制较小。相反，无论模板链上对应的核苷酸是什么，带有3‘-末端A的引物扩增效率都较低。因此，带有标签聚合酶的等位基因特异性聚合酶对与引物的S末端T、G或C的错配提供了最大的模板识别率(40-100倍)，而不是A。倒数第二位的核苷酸造成的错配识别率大约是前者的五分之一(8-20倍)，当T，特别是A占据这个引物位时，扩增效率会降低。因此，我们定义了一些条件，允许对单核苷酸多态(SNPs)的聚合酶链式反应(PCR)介导的分析进行稳健的区分，并降低锚定连接PCR产物的复杂性。(C)2000年学术出版社。
We investigated the effect of primer-template mismatch on the efficiency of polymerase chain reaction. For primers with T, C, or G as the 3' nucleotide, Thermus aquaticus (Taq) DNA polymerase was highly specific for template complementarity to this base, but was somewhat less constrained opposite the penultimate nucleotide. In contrast, primers with a 3'-terminal A were less efficiently amplified regardless of the corresponding nucleotide on the template strand. Thus, allele-specific PCR with Tag polymerase offers the greatest template discrimination (40- to 100-fold) against mismatch to a primer's S'-terminal T, G, or C, but not A. Nucleotides at the penultimate position are responsible for roughly one-fifth as much mismatch discrimination (8- to 20-fold), and amplification efficiency is reduced when T and especially A occupy this primer position. We thus have defined conditions which allow robust discrimination for PCR-mediated analysis of single-nucleotide polymorphisms (SNPs), and for reduction in complexity of anchor-ligation PCR products. (C) 2000 Academic Press.