In situ detection of sequence-specific DNA binding activity specified by a recombinant bacteriophage.

C. R. Vinson, K. L. LaMarco, P. F. Johnson, W. H. Landschulz, S. L. McKnight

Research output: Contribution to journalArticle

333 Scopus citations

Abstract

We have used a recombinant bacteriophage that expresses the DNA-binding domain of C/EBP to optimize conditions for a screening technique that may facilitate the cloning of genes that encode sequence-specific DNA-binding proteins. The method relies on the expression of cDNA inserts in bacteriophage lambda gt11. Fusion protein adsorbed onto nitrocellulose filters is probed with radioactive, double-stranded DNA as a ligand. Two procedures greatly increase the level of binding between ligand and recombinant fusion protein. First, nitrocellulose filters are processed through a denaturation/renaturation regimen using 6 M guanidine hydrochloride. Second, synthetic DNA corresponding to the specific binding site is catenated extensively using DNA ligase. The combination of these procedures leads to remarkably strong detection signals. Specific DNA-binding signals can be detected on duplicate filters, and filters can be washed and reused by repeating the cycle of denaturation/renaturation.

Original languageEnglish (US)
Pages (from-to)801-806
Number of pages6
JournalGenes & development
Volume2
Issue number7
DOIs
StatePublished - Jul 1988

ASJC Scopus subject areas

  • Genetics
  • Developmental Biology

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