A nonradioactive screening method for cloning genes encoding sequence-specific DNA binding proteins.
We have developed a novel nonradioactive screening method for cloning genes encoding sequence-specific DNA binding proteins. This method is derived from previously described protocols developed for the same purpose by using radioactively labeled DNA probes containing protein recognition sequences. This nonradioactive strategy relies upon the use of a small hapten, digoxigenin. Fusion proteins expressed from the recombinant bacteriophage lambda gt11/lambda ZAP are immobilized on nitrocellulose filters and probed with digoxigenin-labeled double-stranded DNA as a ligand. The specifically bound DNA probes can be detected through sequential incubations with antibody-enzyme conjugate and enzyme substrates. This technique has been successfully utilized to isolate several cDNA clones encoding DNA binding proteins.
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PRI/DynCorp, NCI-Frederick Cancer Research and Development Center, Maryland 21702.
D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D004076:Digoxigenin / Q000378:metabolism; D005822:Genetic Vectors; D015698:Genomic Library; D008024:Ligands; D008969:Molecular Sequence Data; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic
D015342:DNA Probes; D004268:DNA-Binding Proteins; D008024:Ligands; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; D004247:DNA; D004076:Digoxigenin