Rapid size determination of mRNAs complementary to cloned DNA sequences: plaque and colony hybrid-selection of cDNAs.
We describe a novel screening method for the rapid size determination of mRNAs. This method has been used successfully to identify the cDNA and genomic DNA clones that had been constructed using the plasmid vector pBR322 or phage vectors M13mp7 and lambda EMBL3. Poly(A) RNA is reverse-transcribed into 32P-labeled cDNA under conditions that yield a high proportion of full-length cDNA copies. This radioactive cDNA is then hybridized in situ to bacterial colonies or phage plaques harboring recombinant DNA molecules. Colonies or plaques containing sequences complementary to abundant or moderately abundant mRNAs are detected by autoradiography and excised from nitrocellulose filters. The hybridized cDNA is eluted from the filter by alkaline denaturation and sized directly by alkaline agarose gel electrophoresis. The mRNAs characterized thus far by this technique measure between 450 and 2100 nucleotides and account for 0.03% to 15% of the mass of cytoplasmic poly(A) RNA.
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D005822:Genetic Vectors; D007739:L Cells / Q000032:analysis; D008099:Liver / Q000032:analysis; D051379:Mice; D008805:Mice, Inbred A; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D010306:Parotid Gland / Q000032:analysis; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism
D012333:RNA, Messenger; D004247:DNA; D012194:RNA-Directed DNA Polymerase