Site-selective blocking of PCR by a caged nucleotide leading to direct creation of desired sticky ends in the products.
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.
Tanaka
Keita
K
Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
Katada
Hitoshi
H
Shigi
Narumi
N
Kuzuya
Akinori
A
Komiyama
Makoto
M
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
D011119:Polynucleotides; D004259:DNA-Directed DNA Polymerase