Selective enrichment of a large size genomic DNA fragment by affinity capture: an approach for genome mapping.
A method to enrich large size DNA fragments obtained by digestion with rare cutting restriction endonucleases was developed and applied for the isolation of a 150 kb SfiI fragment containing the beta-globin gene cluster. The digested DNA is rendered single stranded at the ends by diffusing a strand specific exonuclease into an agarose plug containing DNA. The plug is melted and solution hybridization is then performed with a bridge RNA containing specific sequences from the end of a desired fragment linked to a common probe sequence. The common probe sequence is annealed to a biotinylated RNA and the resulting tripartite hybrid is retained onto a solid matrix containing avidin and specifically released by ribonuclease action. Enrichments of greater than 350 fold have been achieved consistently. Such directed purification of large DNA fragments without cloning can considerably expedite mapping and gene localization in a complex genome and facilitate the construction of sublibraries from defined regions of the genome.
Kandpal
R P
RP
Department of Human Genetics, Yale University School of Medicine, New Haven, CT 06510.
Ward
D C
DC
Weissman
S M
SM
D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D002874:Chromosome Mapping; D004247:DNA / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific; D004586:Electrophoresis / Q000379:methods; D005796:Genes; D015894:Genome, Human; D005914:Globins / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods; D014158:Transcription, Genetic
D005914:Globins; D004247:DNA; C041749:endodeoxyribonuclease SfiI; D015252:Deoxyribonucleases, Type II Site-Specific