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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• 作者：Akinori Kuzuya ; Fuminori Okada ; Makoto Komiyama
• 刊名：Bioconjugate Chemistry
• 出版年：2009
• 出版时间：October 21, 2009
• 年：2009
• 卷：20
• 期：10
• 页码：1924-1929
• 全文大小：295K
• 年卷期：v.20,no.10(October 21, 2009)
• ISSN：1520-4812

文摘

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 nucleotide 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.