Precise site-selective termination of DNA replication by caging the 3-position of thymidine and its application to polymerase chain reaction.
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.
Kuzuya
Akinori
A
Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, Japan.
Okada
Fuminori
F
Komiyama
Makoto
M
D000818:Animals; D017931:DNA Primers / Q000737:chemistry* / Q000235:genetics*; D004261:DNA Replication; D004262:DNA Restriction Enzymes / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D009711:Nucleotides / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D017421:Sequence Analysis; D013936:Thymidine / Q000737:chemistry / Q000235:genetics
D017931:DNA Primers; D009711:Nucleotides; D049452:Green Fluorescent Proteins; D004259:DNA-Directed DNA Polymerase; D004262:DNA Restriction Enzymes; D013936:Thymidine