Restriction enzyme treatment/ligation independent cloning using caged primers for PCR.
We report here a new simple scheme for constructing recombinant vectors that does not require any restriction enzyme, ligase, or any other special enzyme treatment. By using caged primers in PCR, unnatural sticky-ends of any length and sequence are directly prepared on the product after a brief UVA irradiation. Target genes and vectors amplified by this light-assisted cohesive-ending (LACE) PCR join together in desired arrangement, tightly enough to be repaired and ligated in competent cells. We have successfully constructed a recombinant vector based on pBR322 and coding GFP gene by applying this simple and effective system.
Kuzuya
Akinori
A
Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo 153-8904, Japan. kuzu@mkomi.rcast.u-tokyo.ac.jp
Tanaka
Keita
K
Katada
Hitoshi
H
Komiyama
Makoto
M
D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers / Q000737:chemistry*; D004262:DNA Restriction Enzymes; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D049452:Green Fluorescent Proteins; D004262:DNA Restriction Enzymes