Efficient cloning method that selects the recombinant clones.
Directional cloning using cohesive ends is the most efficient cloning method. However, sometimes it is necessary to use blunt ends to clone a DNA fragment into the plasmid vector. Compared with that of cohesive ends, efficiency of blunt-end ligation is low. Compared with the native blunt ends (e.g., SmaI or EcoRV), blunt-end ligation is particularly difficult when blunt ends are derived from overhangs. This results in low efficiency of insertion and high background from self-ligation of the vector. To remedy the problem, we developed a "positive selector" cloning strategy that provides positive selection for the recombinant clones. It is particularly useful when making complex recombinant constructs and the choice of restriction sites is limited.
Loukianov
E
E
University of Cincinnati, College of Medicine, OH, USA.
Loukianova
T
T
Periasamy
M
M
D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000032:analysis; D004262:DNA Restriction Enzymes; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics*
D011994:Recombinant Proteins; D004247:DNA; D004262:DNA Restriction Enzymes