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Nucleic Acids Research, 1981, Vol. 9, No. 10 2251-2266
© 1981

MOLECULAR BIOLOGY

5'-Terminal sequences of eucaryotic mRNA can be cloned with high efficiency

Hartmut Land, Manuel Grez, Hansjörg Hauser+, Werner Lindenmaier+ and Günther Schütz

Institut für Zell- und Tumorbiologie, Deutsches Krebsforschungszentrum Im Neuenheimer Feld 280, D-6900 Heidelberg, GFR

+Present address: GBF, Abteilung Genetik, Mascheroder Weg 1, D-3300 Braunschweig, F.R.G.

Received March 27, 1981. 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 SI 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 103 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.


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