High-efficiency cloning of DNA sequences complementary to mouse neuroblastoma polyadenylated RNA.
A cDNA library was efficiently synthesized from mouse neuroblastoma poly(A)+RNA. Several modifications of the oligo(dC)(dG) tailing procedure were used. After first strand synthesis, a dATP tail was added to the 3'-end of the cDNA. The second strand was primed for synthesis with oligo(dT). Blunt ends were produced on the cDNA by treatment with S1 nuclease. Size-enriched fractions of high molecular weight DNAs were obtained by passing the cDNA over a Sepharose CL-4B column. The optimal tailing time for each cDNA fraction was individually tested. Tailing reactions used terminal deoxynucleotidyl transferase and annealing reactions used a (G)-tailed Pst I cut pBR322. E. coli K12 RR1 cells were transformed and 2.5-5 X 10(6) transformants per microgram cDNA insert were obtained for each size fraction. The transformants had an average insert size of 1200 base pairs and were 98% ampicillin sensitive. Our modifications in the method for cDNA library synthesis had 3 advantages. (1) Homopolymer-primed cDNA treated with S1 nuclease allowed the blunt ends to be tailed synchronously. This allowed a higher transformation efficiency without loss of 5'-sequences. (2) Time tailing determined the most efficient tail length and optimized the transformation efficiency in each size fraction. (3) A Sephadex G-50 mini-column was used to desalt and dry nitrogen was used to concentrate the ds cDNA instead of the usual ethanol precipitation. This resulted in almost 100% recovery of synthesized products at each step of this procedure.
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D000818:Animals; D001482:Base Composition; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics*; D004274:DNA, Recombinant; D004277:DNA, Single-Stranded / Q000235:genetics; D004720:Endonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D051379:Mice; D008970:Molecular Weight; D009447:Neuroblastoma / Q000235:genetics*; D011061:Poly A / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014158:Transcription, Genetic
D004274:DNA, Recombinant; D004277:DNA, Single-Stranded; D012333:RNA, Messenger; D011061:Poly A; D004247:DNA; D004720:Endonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases