Enzyme treatment-free and ligation-independent cloning using caged primers in polymerase chain reactions.
A new simple scheme for constructing recombinant vectors that does not require any restriction enzyme, ligase, or any other special enzyme treatment has been developed. By using caged primers in PCR, unnatural sticky-ends of any sequence, which are sufficiently long for ligation-independent cloning (LIC), 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 the desired arrangement in a simple mixture of them, tightly enough to be repaired and ligated in competent cells.
Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904, Japan. kuzuya@kansai-u.ac.jp
Akinori
A
Kuzuya
Keita
K
Tanaka
Hitoshi
H
Katada
Makoto
M
Komiyama
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D053904:DNA Breaks, Single-Stranded; D017931:DNA Primers / Q000138:chemical synthesis*; D004260:DNA Repair; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000138:chemical synthesis / Q000528:radiation effects; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014466:Ultraviolet Rays
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins