A General Method of Gene Isolation

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RESUMO

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° (50% formamide, 0.3 M NaCl-0.03 M Na3 citrate) to allow efficient DNA-RNA interaction in the stationary phase, while unreacted and any reassociated DNA is denatured at 90° and then recirculated into the hybridization chamber. Between 40 and 45% of fragmented 32P-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 104-fold excess of sheared E. coli DNA, nearly half of the [32P]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.

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