Enrichment of specific genes from genomic DNA or from clone library DNA, using R-looping.
We have developed a procedure for enriching DNA for specific sequences that is based on R-looping (Thomas et al., 1976). R-loops are formed with the DNA using mRNAs containing the sequence of interest and then isolated on poly(U)-sepharose via the poly(A) tail of the mRNA. Model experiments showed that plasmid DNA containing a cDNA copy of an immunoglobulin kappa chain mRNA could be selectively retrieved using this procedure. Approx. 5-10% of the kappa sequences in mouse embryo DNA could be recovered by R-looping, while non-specific binding of mouse DNA to the poly(U)-sepharose column was 0.03-0.04%. This represents a 100-200-fold enrichment of mouse genomic kappa sequences. We have also used the procedure to rapidly screen a mouse clone library for immunoglobulin heavy chain genes. DNA from the clone library was enriched 100-200-fold using immunoglobulin heavy chain mRNAs, and the enriched DNA repackaged in vitro to recover the phage.
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D002846:Chromatography, Affinity; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005822:Genetic Vectors; D007147:Immunoglobulin Light Chains / Q000235:genetics*; D007145:Immunoglobulin kappa-Chains / Q000235:genetics*; D051379:Mice; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger
D004274:DNA, Recombinant; D007147:Immunoglobulin Light Chains; D007145:Immunoglobulin kappa-Chains; D012333:RNA, Messenger