Unit

UNIT 12.11 Determination of Protein-DNA Sequence Specificity by PCR-Assisted Binding-Site Selection

  1. Roy M. Pollock

Published Online: 1 MAY 2001

DOI: 10.1002/0471142727.mb1211s33

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Pollock, R. M. 2001. Determination of Protein-DNA Sequence Specificity by PCR-Assisted Binding-Site Selection. Current Protocols in Molecular Biology. 33:12.11:12.11.1–12.11.11.

Author Information

  1. Ariad Pharmaceuticals, Inc., Cambridge, Massachusetts

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: JAN 1996

Abstract

Binding-site selection is used to determine the target specificity of a sequence-specific DNA-binding protein. In this unit, a pool of random-sequence oligonucleotides is used as the source of potential binding sites. This pool is incubated with extract containing the DNA-binding protein of interest and the protein-DNA complexes are isolated by immunoprecipitation with an antibody specific for the protein under investigation. Unbound oligonucleotides are removed by gentle washing, and bound oligonucleotides are recovered, amplified by the polymerase chain reaction (PCR), and used as input DNA for a further round of binding, recovery, and amplification. After four rounds of selection, progress of the procedure is monitored by mobility shift analysis of the selected oligonucleotide pools. In the Support Protocol, individual binding sites are isolated from the appropriate complex on a mobility shift gel, cloned into plasmids, and examined by sequencing.