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Differential cloning using in‐gel competitive reassociation
Author(s) -
Yokota Hiroshi,
Amano Shinya,
Yamane Tetsuo,
Ataka Kazuto,
Kikuya Eriko,
Oishi Michio
Publication year - 1995
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150160145
Subject(s) - dna , cloning (programming) , biology , biotinylation , genome , genomic dna , sequencing by ligation , molecular cloning , polymerase chain reaction , microbiology and biotechnology , gel electrophoresis , restriction fragment , genetics , genomic library , computational biology , gene , complementary dna , base sequence , computer science , programming language
We describe the principle and actual processes of a differential cloning procedure designed for cloning of anonymous restriction DNA fragments whose molecular sizes differ between two genomic DNA preparations from higher organisms as a result of DNA rearrangement, polymorphism, etc. The procedure, which was extensively modified from the original one and still employs in‐gel competitive reassociation (IGCR) as the basic principle, aims for cloning of DNA fragments which exist in one copy or less per mammalian genome. The modified procedure consists of dissociation and reassociation of biotinylated restriction digests of target DNA fragments (from which clones are to be isolated) in the presence of a large excess of reference (competitor) DNA in gel after electrophoresis, which is followed by absorption of the target DNA fragments to streptavidin‐coated tubes and solid‐phase polymerase chain reaction. After repeating these steps we attained substantial enrichment of altered DNA fragments which were originally present in one copy or less per complex mammalian genome.