Systematic characterization of 2'-deoxynucleoside- 5'-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on enzymatic production of modified DNA.
Systematic characterization of 2'-deoxynucleoside- 5'-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on enzymatic production of modified DNA.
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DOI：
10.1093/nar/gkl637
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发表时间：
2006
影响因子：
14.9
通讯作者：
Sawai H
中科院分区：
文献类型：
--
作者：
Kuwahara M;Nagashima J;Hasegawa M;Tamura T;Kitagata R;Hanawa K;Hososhima S;Kasamatsu T;Ozaki H;Sawai H
我们合成了2′-脱氧尿苷三磷酸和2′-脱氧胞苷三磷酸的C5修饰类似物，并研究了它们作为PCR底物的作用，使用Taq、Tth、Vent（exo-）、KOD Dash和KOD（exo-）聚合酶和pUC 18质粒DNA作为模板。这些测定在具有不同T/C含量的pUC 18的两个不同扩增区域上进行，所述不同T/C含量预期对于修饰的dU或dC的掺入具有相对高的屏障。基于260种不同的检测（26种修饰的三磷酸酶× 5种DNA聚合酶× 2个扩增区），似乎全长PCR产物的产生不仅取决于取代的化学结构和聚合酶的性质，而且还取决于取代是在dU还是dC上。此外，模板序列极大地影响PCR产物的产生，这取决于DNA聚合酶和修饰的三磷酸的组合。通过使用含有C5-修饰的dU的引物和模板检测引物延伸反应，我们发现在延伸链的3′端的修饰的dU极大地影响DNA聚合酶的催化效率，而在模板链上的延伸位点对面的修饰的dU对催化效率的影响较小。
We synthesized C5-modified analogs of 2′-deoxyuridine triphosphate and 2′-deoxycytidine triphosphate and investigated them as substrates for PCRs using Taq, Tth, Vent(exo-), KOD Dash and KOD(exo-) polymerases and pUC 18 plasmid DNA as a template. These assays were performed on two different amplifying regions of pUC18 with different T/C contents that are expected to have relatively high barriers for incorporation of either modified dU or dC. On the basis of 260 different assays (26 modified triphosphates × 5 DNA polymerases × 2 amplifying regions), it appears that generation of the full-length PCR product depends not only on the chemical structures of the substitution and the nature of the polymerase but also on whether the substitution is on dU or dC. Furthermore, the template sequence greatly affected generation of the PCR product, depending on the combination of the DNA polymerase and modified triphosphate. By examining primer extension reactions using primers and templates containing C5-modified dUs, we found that a modified dU at the 3′ end of the elongation strand greatly affects the catalytic efficiency of DNA polymerases, whereas a modified dU opposite the elongation site on the template strand has less of an influence on the catalytic efficiency.
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影响因子：
46.9
作者：
Breaker, RR
通讯作者：
Breaker, RR
影响因子：
5.6
作者：
Hashimoto, H;Nishioka, M;Kai, Y
通讯作者：
Kai, Y
影响因子：
4.8
作者：
Kim, TW;Brieba, LG;Kool, ET
通讯作者：
Kool, ET
影响因子：
14.9
作者：
Gourlain, T;Sidorov, A;Williams, DM
通讯作者：
Williams, DM
影响因子：
16.6
作者：
Jäger, S;Famulok, M
通讯作者：
Famulok, M