Use of DNA-protein interaction to isolate specific genomic DNA sequences.
We describe a simple and rapid method for the isolation of specific genomic DNA sequences recognized by DNA-binding proteins. This procedure consists of four steps: (1) restriction enzyme digestion and size fractionation of genomic DNA; (2) DNA--protein binding using the gel mobility-shift assay; (3) ligation of isolated DNA fragments followed by transformation of Escherichia coli; and (4) screening of recombinant clones for inserts containing specific DNA--protein binding sequences. We have used this protocol to isolate human DNA sequences, 100-200 bp in size, that are recognized by both partially purified and affinity purified proteins. Unlike other procedures designed to identify genomic target sequences, the method described does not require polymerase chain reaction or successive immunoprecipitations.
Hobson
K
K
Queensland Institute of Medical Research, Herston, Brisbane, Australia.
Singh
S P
SP
Lavin
M F
MF
D001260:Ataxia Telangiectasia / Q000235:genetics; D002461:Cell Line, Transformed; D004247:DNA / Q000302:isolation & purification*; D004251:DNA Transposable Elements; D004274:DNA, Recombinant / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004854:Herpesvirus 4, Human; D006801:Humans; D010957:Plasmids
D004251:DNA Transposable Elements; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D004247:DNA