Use of RecA protein to enrich for homologous genes in a genomic library.
RecA protein-coated probe has been utilized to enrich genomic digests for desired genes in order to facilitate cloning from genomic libraries. Using a previously cloned HLA-B27 gene as the recA-coated enrichment probe, we obtained a mean 108x increase in the ratio of specific to nonspecific plaques in lambda libraries screened for B27 variant alleles of estimated 99% homology to the probe. Class I genes of lesser homology were less enriched: 6.7x for non-B27 genes of estimated greater than 95% homology and 3.7x for other-Class I genes of greater than 80% homology. Loss of genomic DNA during the enrichment procedure can, however, restrict application of this technique whenever starting genomic DNA is very limited. Nevertheless, the impressive reduction in cloning effort and material makes recA enrichment a useful new tool for cloning homologous genes from genomic DNA.
D000483:Alleles; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004274:DNA, Recombinant / Q000302:isolation & purification; D014644:Genetic Variation; D006680:HLA Antigens / Q000235:genetics*; D015796:HLA-B27 Antigen; D006801:Humans; D012150:Polymorphism, Restriction Fragment Length; D011938:Rec A Recombinases / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid
D004274:DNA, Recombinant; D006680:HLA Antigens; D015796:HLA-B27 Antigen; D011938:Rec A Recombinases