Retrieval of human DNA from rodent-human genomic libraries by a recombination process |
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Authors: | R L Neve G A Bruns T P Dryja D M Kurnit |
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Institution: | 1. Division of Clinical Genetics, Children''s Hospital Medical Center, Boston, MA 02115 USA Tel. (617)-735-7240;2. Department of Ophthalmology, Massachusetts Eye and Ear Hospital, Boston, MA 02114 USA Tel. (617) 523-7900;3. Division of Genetics, Brigham and Women''s Hospital, Boston, MA 02115 U.S.A. Tel. (617) 732-2296 |
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Abstract: | Human Alu repeat ("BLUR") sequences have been cloned into the mini-plasmid vector piVX. The resulting piBLUR clones have been used to rescue selectively, by recombination, bacteriophage carrying human DNA sequences from genomic libraries constructed using DNA from rodent-human somatic cell hybrids. piBLUR clones are able to retrieve human clones from such libraries because at least one Alu family repeat is present on most 15 to 20 kb fragments of human DNA and because of the relative species-specificity of the sequences comprising the Alu family. The rapid, selective plaque purification achieved results in the construction of a collection of recombinant phage carrying diverse human DNA inserts from a specific subset of the human karyotype. Subfragments of two recombinants rescued from a mouse-human somatic cell hybrid containing human chromosomes X, 10, 13, and 22 were mapped to human chromosomes X and 13, respectively, demonstrating the utility of this protocol for the isolation of human chromosome-specific DNA sequences from appropriate somatic cell hybrids. |
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Keywords: | Recombinant DNA Alu family repeats BLUR lambda Charon phage vectors human gene mapping X chromosome Alu family human interspersed family of repeated sequences BLUR bp base pairs EtBr ethidium bromide IPTG kb kilobase pairs pfu plaque-forming units see Neve and Kurnit (1983) MATERIALS AND METHODS section c SDS sodium dodecyl sulfate X-gal [ ] indicates plasmid-carrier state |
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