Multiple bandshift assay: rapid identification and cloning of DNA fragments containing specific protein-binding sites.
A new rapid method for the identification and cloning of DNA fragments containing specific protein-binding domains is based on the common bandshift assay. Cloned DNA is digested with a restriction endonuclease recognizing a particular 4-bp sequence, an aliquot of this digest is end-labelled and used in protein binding reactions with and without protein extract. The binding reactions are then loaded onto nondenaturing polyacrylamide gel. The main portion of the digest is run in a parallel lane and serves as a source of fragments for cloning. Autoradiography of the wet gel reveals loss in intensity of some bands from the restriction digest incubated with the protein extract. DNA fragments corresponding to these bands are cut out from the gel; DNA is eluted and cloned in the M13 vector, thus allowing rapid and simple sequencing of the inserts. The method, termed multiple bandshift assay, is especially useful when screening relatively long DNA fragments (of several kb) for potential protein-binding domains. The procedure was used to study interaction of HeLa-cell nuclear proteins with a 5.2-kb downstream region of pseudorabies virus immediate-early gene (C. Vlcek, Z. Kozmík, V. Paces, S. Schirm and M. Schwyzer, unpublished).
Kozmík
Z
Z
Institute of Molecular Genetics, Prague, Czechoslovakia.
Paces
V
V
D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000032:analysis*; D003850:Deoxyribonuclease I; D005814:Genes, Viral; D006367:HeLa Cells; D006801:Humans; D017874:Immediate-Early Proteins; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D014764:Viral Proteins / Q000235:genetics
D004274:DNA, Recombinant; D017874:Immediate-Early Proteins; D009687:Nuclear Proteins; D014764:Viral Proteins; C047583:pseudorabies virus immediate early proteins; D003850:Deoxyribonuclease I