Work

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

Year: 1988

Type: preprint

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.

Source: Genes & Development

DOI: 10.1101/gad.2.7.801

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

Institutions: Carnegie Institution for Science, Department of Embryology, Carnegie Observatories

Language: English

Topic: Genomics and Chromatin Dynamics

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: bronze

Funders: American Cancer Society, Carnegie Corporation of New York, Damon Runyon Cancer Research Foundation, National Institutes of Health

OpenAlex ID: W2072276788

Cites: 26

Cited by: 492

Related to: 10