5'-Terminal sequences of eucaryotic mRNA can be cloned with high efficiency.
A method for cloning mRNAs has been used which results in a high yield of recombinants containing complete 5'-terminal mRNA sequences. It is not dependent on self-priming to generate double-stranded DNA and therefore the S1 nuclease digestion step is not required. Instead, the cDNA is dCMP-tailed at its 3'-end with terminal deoxynucleotidyl transferase (TdT). The synthesis of the second strand is primed by oligo(dG) hybridized to the 3'-tail. Double-stranded cDNA is subsequently tailed with dCTP and annealed to dGMP-tailed vector DNA. This approach overcomes the loss of the 5'-terminal mRNA sequences and the problem of artifacts which may be introduced into cloned cDNA sequences. Chicken lysozyme cDNA was cloned into pBR322 by this procedure with a transformation efficiency of 5 x 10(3) recombinant clones per ng of ds-cDNA. Sequence analysis revealed that at least nine out of nineteen randomly isolated plasmids contained the entire 5'-untranslated mRNA sequence. The data strongly support the conclusion that the 5'-untranslated region of the lysozyme mRNA is heterogeneous in length.
D009189:Avian Myeloblastosis Virus / Q000201:enzymology; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000378:metabolism*; D009113:Muramidase / Q000235:genetics; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase
D004274:DNA, Recombinant; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase; D009113:Muramidase