Portal de Periódicos da CAPES

A method to identify and characterize Z-DNA binding proteins using a linear oligodeoxynucleotide

1993; Oxford University Press; Volume: 21; Issue: 11 Linguagem: Inglês

10.1093/nar/21.11.2669

1362-4962

Alan Herbert, Alexander Rich,

Advanced biosensing and bioanalysis techniques

An oligodeoxynucleotide that readily flips to the Z-DNA conformation in 10mM MgCl2 was produced by using Klenow enzyme to incorporate 5-bromodeoxycytosine and deoxyguanosine into a (dC-dG)22 template. During synthesis the oligomer can be labeled with 32P to high specific activity. The labeled oligodeoxynucleotide can be used in bandshift experiment to detect proteins that bind Z-DNA. This allows the binding specificity of such proteins to be determined with high reliability using unlabeled linear and supercoiled DNA competitors. In addition, because the radioactive oligodeoxynucleotide contains bromine atoms, DNA-protein complexes can be readily crosslinked using UV light. This allows an estimate to be made of the molecular weight of the proteins that bind to the radioactive probe. Both techniques are demonstrated using a goat polyclonal anti-Z-DNA antiserum.