A universal method for directional cloning of PCR products based on asymmetric PCR.
We have developed a novel protocol for directional cloning of PCR products into any vector. The target sequence is amplified in two parallel asymmetric PCRs using specially but simply designed primers. Two single-stranded products are produced and they are annealed to form a double-stranded DNA fragment bearing overhangs at both ends that correspond to the restriction overhangs of certain restriction enzymes. The fragment can then be cloned into a certain vector previously treated with the corresponding enzymes without restriction of the inserted fragment. Compared with previously published protocols, the procedure described in this paper is highly efficient and it is independent of the restriction sites of the insert and is therefore applicable to molecular biology and biotechnology studies.
Wang
Bao-Li
BL
Key Research Laboratory of Hormone and Development Affiliated to the Ministry of Health, Institute of Endocrinology, Tianjin Medical University, Tianjin, People's Republic of China. bliwang72@yahoo.com.cn
Jiao
Yan-Li
YL
Li
Xiao-Xia
XX
Zheng
Fang
F
Liang
Hui
H
Sun
Zi-Yang
ZY
Guo
Gang
G
D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D051381:Rats
D017931:DNA Primers; D008565:Membrane Proteins; D004262:DNA Restriction Enzymes