A generally applicable improved method for preparation of single stranded cDNA probes from clones constructed in M13...

A generally applicable simplified procedure for the preparation of radiolabeled cDNA hybridization probes from cDNA clones in M13 (M13mp8) bacteriophage vectors is described. A cDNA copy of the insert DNA is synthesized by controlled reaction with the Klenow fragment of E. coli DNA polymerase I, primed with oligo-dT or sequencing primer. The cDNA is separated from the recombinant phage DNA template by alkaline gel electrophoresis. Sensitivity of the cDNAs was tested by quantitative measurement of specific mRNAs in solution hybridization under RNA (R0t analysis) or cDNA (RNA titration) excess conditions. The procedure permits measurement of mRNA levels as small as 0.00001-0.00006% in total RNA preparation. Cellular accumulation of hormone-induced mRNAs for the milk proteins, whey acidic protein and epsilon-casein was also measured using the cDNAs.