Open AccessEfficient Cloning Method that Selects the Recombinant Clones
Author(s) -
Evgeny Loukianov,
Tanya Loukianova,
Muthu Periasamy
Publication year - 1997
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/97232st05
Subject(s) - recombinant dna , ligation , cloning (programming) , ecorv , plasmid , molecular cloning , sticky and blunt ends , clone (java method) , biology , restriction enzyme , cloning vector , multiple cloning site , vector (molecular biology) , in vitro recombination , genetics , microbiology and biotechnology , computational biology , dna , computer science , ecori , gene , gene expression , programming language
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 Eco RV), 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.