title: Site-selective blocking of PCR by a caged nucleotide leading to direct creation of desired sticky ends in the products.
abstract: In order to terminate the polymerase reaction at a desired position, a caged thymine derivative--4-O-[2-(2-nitrophenyl)propyl]thymine--was incorporated into PCR primers. In the PCR cycles, the elongation of the nascent strand (5'-->3' direction) by polymerase was site-selectively terminated at the 3'-side of T(NPP). Accordingly, predetermined protruding ends were obtained after the removal of the protecting group by short UVA irradiation. Recombinant vectors coding the GFP gene were successfully prepared by direct ligation of these light-assisted cohesive-ending PCR (LACE-PCR) products with scission fragments obtained by use either of restriction enzymes or of artificial restriction DNA cutters and were used for transformation of E. coli.
authors.affiliation: Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
authors.forename: Keita
authors.initials: K
authors.lastname: Tanaka
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Katada
authors.forename: Narumi
authors.initials: N
authors.lastname: Shigi
authors.forename: Akinori
authors.initials: A
authors.lastname: Kuzuya
authors.forename: Makoto
authors.initials: M
authors.lastname: Komiyama
mesh_terms: D001483:Base Sequence; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008027:Light; D016133:Polymerase Chain Reaction / Q000379:methods*; D011119:Polynucleotides / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D011119:Polynucleotides; D004259:DNA-Directed DNA Polymerase