Direct preparation of sticky-ended duplexes within PCR by using caged primers.
Control of the terminal structures of PCR products is crucially important to facilitate molecular biology and biotechnology. Here, we report a new method to prepare PCR products having desired sticky ends directly after thermal cycles. When a pair of caged primers is used, polymerase reaction is site-selectively terminated in front of the caged nucleotide, and the 5'- portion of the primer remains single-stranded throughout the reaction. Successive removal of the photo-cleavable protecting group gives restriction-enzyme free sticky ends on the product. We succeeded in applying this technique to make a recombinant vector bearing GFP gene.
Tanaka
Keita
K
RCAST, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904, Japan.
Katada
Hitoshi
H
Shigi
Narumi
N
Kuzuya
Akinori
A
Komiyama
Makoto
M
D017931:DNA Primers / Q000737:chemistry* / Q000528:radiation effects; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D014466:Ultraviolet Rays
D017931:DNA Primers; D049452:Green Fluorescent Proteins