Elsevier

Gene

Volume 10, Issue 2, July 1980, Pages 147-155
Gene

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

https://doi.org/10.1016/0378-1119(80)90132-8Get rights and content

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.

References (34)

  • N. Sternberg et al.

    In vitro packaging of a λDam vector containing EcoRI DNA fragments of Escherichia coli and phage P1

    Gene

    (1977)
  • D.C. Tiemeier et al.

    Purification and cloning of a mouse ribosomal gene fragment in coliphage lambda

    Gene

    (1977)
  • J.M. Adams et al.

    Molecular cloning of mouse immunoglobulin heavy chain mRNAs coding for μ, α, γ1, γ2a and γ3 chains

    Biochemistry

    (1980)
  • W.D. Benton et al.

    Screening λgt recombinant clones by hybridisation to single plaques in situ

    Science

    (1977)
  • F.R. Blattner et al.

    Charon phages: safer derivatives of bacteriophage lambda for DNA cloning

    Science

    (1977)
  • D.D. Brown et al.

    Methods of gene isolation

    Annu. Rev. Biochem.

    (1974)
  • J.R. Cameron et al.

    In vitro construction of bacteriophage λ carrying segments of the Escherichia coli chromosome: selection of hybrids containing the gene for DNA ligase

  • View full text