Preparation of long sticky ends for universal ligation-independent cloning: sequential T4 DNA polymerase treatments.
Ligation-independent cloning (LIC) is a useful method for efficient directional cloning of a PCR product. LIC requires a specially designed vector containing a long stretch of sequence that is missing any one of the four nucleotides. When the linearized vector is treated with T4 DNA polymerase, in the presence of the absent base, long single-stranded overhangs are generated that are suitable for cloning. In this study, long and efficient sticky ends for LIC were produced by sequential T4 DNA polymerase treatments at non-specific sequences on a commercially available vector. All restriction enzyme sites become available in the current LIC.
Tachibana
Akira
A
Department of Bioengineering, Graduate School of Engineering, Osaka City University, Sugimoto 3-3-138, Sumiyoshi-ku, Osaka 558-8585, Japan. tatibana@bioa.eng.osaka-cu.ac.jp
Tohiguchi
Kazuo
K
Ueno
Takayuki
T
Setogawa
Yuichi
Y
Harada
Ayako
A
Tanabe
Toshizumi
T
D003001:Cloning, Molecular / Q000379:methods*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005822:Genetic Vectors; D010957:Plasmids; D014764:Viral Proteins / Q000378:metabolism*
D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; D004259:DNA-Directed DNA Polymerase