Differential cloning using in-gel competitive reassociation.
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.
Yokota
H
H
Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan.
Amano
S
S
Yamane
T
T
Ataka
K
K
Kikuya
E
E
Oishi
M
M
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification* / Q000592:standards; D017931:DNA Primers / Q000235:genetics; D004586:Electrophoresis / Q000379:methods*; D005260:Female; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D051381:Rats; D017208:Rats, Wistar; D017384:Sequence Deletion
D017931:DNA Primers; D004247:DNA