Recombination-based assay (RBA) for screening bacteriophage lambda libraries.
The recombination-based assay represents a convenient way to screen a complex library constructed in bacteriophage lambda for homology to a given sequence cloned into a specially designed plasmid. The technique serves to screen a bacteriophage library rapidly and efficiently with a sequence cloned into a plasmid; counterselection then yields the gene product of interest with its plasmid carrier deleted. Because 10(6) to 10(7) plaque-forming units (pfu) may be screened using several petri dishes, and the homology for crossing-over need only be greater than 25 bp, the RBA represents an efficient way to screen complex lambda libraries rapidly for homology to a given sequence. In this procedure, a lambda library is screened using a specially designed plasmid carrying the desired target sequence. Recombinants arising from cross-over events between the plasmid and a bacteriophage carrying a corresponding region of homology are selected by their ability to grow on strain DM21. Growth of lambda on DM21 requires the presence of an allele encoded on the plasmid to suppress an amber mutation in the host strain that prevents lambda propagation. Recovery of the original phage carrying the target sequence requires a reversal of the homologous recombination event. This reversal occurs spontaneously, and is detected by PCR amplification using primers that flank the cloning site in the lambda vector.
Kurnit
D M
DM
University of Michigan Medical Center, Ann Arbor, Michigan, USA.
D010582:Bacteriophage lambda / Q000235:genetics*; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000145:classification / Q000235:genetics; D015723:Gene Library; D005838:Genotype; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic
D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides