Enrichment of specific genes from genomic DNA or from clone library DNA, using R-looping

Gene. 1980 Jul;10(2):147-55. doi: 10.1016/0378-1119(80)90132-8.

Abstract

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.

Publication types

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

MeSH terms

  • Animals
  • Bacteriophage lambda / genetics
  • Chromatography, Affinity
  • DNA, Recombinant
  • Escherichia coli / genetics
  • Genes
  • Genetic Vectors
  • Immunoglobulin Light Chains / genetics*
  • Immunoglobulin kappa-Chains / genetics*
  • Mice
  • Nucleic Acid Hybridization
  • RNA, Messenger

Substances

  • DNA, Recombinant
  • Immunoglobulin Light Chains
  • Immunoglobulin kappa-Chains
  • RNA, Messenger