Precise Site-Selective Termination of DNA Replication by Caging The 3-Position of Thymidine and Its Application to Polymerase Chain Reaction
Precise Site-Selective Termination of DNA Replication by Caging The 3-Position of Thymidine and Its Application to Polymerase Chain Reaction
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DOI：
10.1021/bc900254e
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发表时间：
2009-10-01
影响因子：
4.7
通讯作者：
Komiyama, Makoto
中科院分区：
文献类型：
--
作者：
Kuzuya, Akinori;Okada, Fuminori;Komiyama, Makoto
合成了一种新的笼状胸苷3-N-（2-（2-硝基苯基）丙氧基甲基）胸苷（T-NPPOM），并将其作为DNA聚合酶反应的位点选择性终止剂用于光辅助粘末端PCR（LACE-PCR），使其在短暂的UVA照射下直接产生粘末端PCR产物。使用涉及T-NPPOM的模板进行的引物延伸实验表明，这种笼状核苷酸有效地和位点选择性地阻断了PCR中常用的各种聚合酶的反应。使用其他先前报道的笼状胸苷观察到的核碱基的错误掺入几乎没有发生。事实证明，笼化基团的轻微结构差异可以显著提高聚合酶反应的终止产率。通过使用含有T-NPPOM的引物制备编码GFP基因的LACE-PCR产物，并将其与使用限制性酶制备的载体片段连接。重组质粒成功转化E.杆菌
A new caged thymidine, 3-N-(2-(2-nitrophenyl)propyloxymethyl)thymidine (T-NPPOM) was synthesized and used as a site-selective terminator of DNA-polymerase reaction in light-assisted cohesive-ending PCR (LACE-PCR), which directly gives sticky-ended PCR products after brief UVA irradiation. Primer-extension experiments using a template involving T-NPPOM have shown that this caged inucleotide efficiently and site-selectively blocks reactions of a variety of polymerases commonly used in PCR Misincorporation of nucleobases, observed with the use of other previously reported caged thymidines, scarcely occurred. It has turned out that a slight structural difference of caging groups can significantly improve the termination yield of polymerase reactions. A LACE-PCR product coding GFP gene was prepared by using primers containing T-NPPOM and was ligated with a vector fragment prepared using restriction enzymes. The resulting recombinant vector successfully transformed E. coli.