EXTENSION OF BASE MISPAIRS BY TAQ DNA-POLYMERASE - IMPLICATIONS FOR SINGLE NUCLEOTIDE DISCRIMINATION IN PCR
EXTENSION OF BASE MISPAIRS BY TAQ DNA-POLYMERASE - IMPLICATIONS FOR SINGLE NUCLEOTIDE DISCRIMINATION IN PCR
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DOI：
10.1093/nar/20.17.4567
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发表时间：
1992-09-11
影响因子：
14.9
通讯作者：
GOODMAN, MF
中科院分区：
文献类型：
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作者：
HUANG, MM;ARNHEIM, N;GOODMAN, MF
使用水生栖热菌（Taq）DNA聚合酶测量模板-引物3 '-末端处匹配和错配碱基对的所有构型的延伸效率。转换错配，A（引物）。C，C。A，G . T和T。G的延伸效率比它们正确配对的对应物低10（-3）至10（-4）倍。T . C和T-T，A-A约为10（-6），G. A、A-G、G-G和C. C小于10（-6）。颠换错对C（引物）。T以高效率延伸，与正确的A.T碱基对相比约为10（-2）。扩展C . T错配不太可能是由引物-模板错配引起的。观察到Taq聚合酶以相似的亲和力与正确配对和错误配对的引物-模板3 '端中的每一个结合。因此，Taq聚合酶不能有效地延伸错配似乎是酶的固有性质，而不是由于不能结合3 '末端错配。对于几乎所有的错配，C。T是例外，Taq聚合酶表现出约100至1000倍更大的歧视，对错配延伸相比，禽成髓细胞瘤逆转录酶和HIV-1逆转录酶，延长大多数错配碱基对许可。在45 ℃、55 ℃和70 ℃下测量Taq聚合酶的相对错配延伸效率，发现其与温度无关。错配延伸数据在设计使用PCR的实验以区分单个核苷酸不同的序列时应该是重要的。
Thermus aquaticus (Taq) DNA polymerase was used to measure the extension efficiency for all configurations of matched and mismatched base pairs at template-primer 3'-termini. The transition mispairs, A(primer) . C, C . A, G . T, and T . G were extended 10(-3) to 10(-4)-fold less efficiently than their correctly paired counterparts. Relative efficiencies for extending transversion mispairs were 10(-4) to 10(-5) for T . C and T-T, about 10(-6) for A-A, and less than 10(-6) for G.A, A-G, G-G and C.C. The transversion mispair C(primer) . T was extended with high efficiency, about 10(-2) compared to a correct A.T basepair. The unexpected ease of extending the C . T mismatch was not likely to have been caused by primer-template misalignment. Taq polymerase was observed to bind with similar affinities to each of the correctly paired and mispaired primer-template 3'-ends. Thus, the failure of Taq polymerase to extend mismatches efficiently appears to be an intrinsic property of the enzyme and not due to an inability to bind to 3'-terminal mispairs. For almost all of the mispairs, C . T being the exception, Taq polymerase exhibits about 100 to 1000-fold greater discrimination against mismatch extension compared to avian myeloblastosis reverse transcriptase and HIV-1 reverse transcriptase which extend most mismatched basepairs permissively. Relative mismatch extension efficiencies for Taq polymerase were measured at 45-degrees-C, 55-degrees-C and 70-degrees-C and found to be independent of temperature. The mispair extension data should be important in designing experiments using PCR to distinguish between sequences that vary by a single nucleotide.