RecA-assisted rapid enrichment of specific clones from model DNA libraries.
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.
Teintze
M
M
Dept. of Medicine, Cornell University Medical College, New York, NY 10021, USA.
Arzimanoglou
I I
II
Lovelace
C I
CI
Xu
Z J
ZJ
Rigas
B
B
D000418:Albumins / Q000235:genetics; D017104:Bacteriophage M13 / Q000235:genetics; D002482:Cellulose / Q000031:analogs & derivatives; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods*; D015342:DNA Probes; D004274:DNA, Recombinant / Q000235:genetics; D004277:DNA, Single-Stranded; D015723:Gene Library; D006801:Humans; D009838:Oligodeoxyribonucleotides; D011938:Rec A Recombinases / Q000378:metabolism*; D012641:Selection, Genetic
D000418:Albumins; D015342:DNA Probes; D004274:DNA, Recombinant; D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; C021722:oligo(dT)-cellulose; D002482:Cellulose; D011938:Rec A Recombinases