A general method of gene isolation.
A general method of gene isolation has been developed that involves the chemical linkage of RNA to cellulose by a water-soluble carbodiimide, and the continuous circulation of DNA containing specific sequences complementary to the RNA. The temperature of the cellulose matrix is maintained at 37 degrees (50% formamide, 0.3 M NaCl-0.03 M Na(3) citrate) to allow efficient DNA-RNA interaction in the stationary phase, while unreacted and any reassociated DNA is denatured at 90 degrees and then recirculated into the hybridization chamber. Between 40 and 45% of fragmented (32)P-labeled simian virus (SV)40 DNA was removed from the circulating solution when cellulosebound SV40-specific RNA, assymmetrically transcribed in vitro with Escherichia coli RNA polymerase, was used. In the presence of 10(4)-fold excess of sheared E. coli DNA, nearly half of the [(32)P]SV40 DNA was recovered from the mixture as a DNA-RNA hybrid with negligible contamination by bacterial DNA. The isolation procedure is almost quantitative for the complementary DNA. The efficiency and selectivity of this method permit the isolation of a defined DNA sequence from a large and complex genome.
D002234:Carbodiimides; D002482:Cellulose / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D004279:DNA, Viral / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli; D005796:Genes; D008722:Methods; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D010759:Phosphorus Isotopes; D012367:RNA, Viral / Q000378:metabolism; D013539:Simian virus 40 / Q000032:analysis* / Q000378:metabolism
D002234:Carbodiimides; D004269:DNA, Bacterial; D004279:DNA, Viral; D010759:Phosphorus Isotopes; D012367:RNA, Viral; D002482:Cellulose