RecA-assisted rapid enrichment of specific clones from model DNA libraries

Biochem Biophys Res Commun. 1995 Jun 26;211(3):804-11. doi: 10.1006/bbrc.1995.1883.

Abstract

An approach to library screening is being developed, in which the desired clone is "fished" out of a mixture of all the recombinants in a library with a RecA-coated probe. In the current embodiment of this method, we used as a probe the (+) strand of an M13 phage containing a fragment of the human albumin gene and a (dA)49 stretch. We screened a library of two plasmids, one containing the same albumin fragment as the probe, and one heterologous to the probe in 50-100 fold molar excess. The plasmids were linearized. Probe and library were reacted in the presence of RecA, the mixture was loaded onto an oligo(dT) column, which retained the probe-target complex by base-pairing to the dAs of the probe, the uncaptured plasmids were washed, and the probe-target complex was released from the column, religated and propagated into E. coli. Recovery of the homologous target was 15-28%, and enrichment for the homologous plasmid was 200 to 400-fold. This approach may provide a general method for expedited DNA library screening.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Albumins / genetics
  • Bacteriophage M13 / genetics
  • Cellulose / analogs & derivatives
  • Chromatography, Affinity
  • Cloning, Molecular / methods*
  • DNA Probes*
  • DNA, Recombinant / genetics
  • DNA, Single-Stranded
  • Gene Library*
  • Humans
  • Oligodeoxyribonucleotides
  • Rec A Recombinases / metabolism*
  • Selection, Genetic*

Substances

  • Albumins
  • DNA Probes
  • DNA, Recombinant
  • DNA, Single-Stranded
  • Oligodeoxyribonucleotides
  • oligo(dT)-cellulose
  • Cellulose
  • Rec A Recombinases