5′-Terminal sequences of eucaryotic mRNA can be cloned with high efficiency (original) (raw)

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Institut für Zell- und Tumorbiologie, Deutsches Krebsforschungszentrum

Im Neuenheimer Feld 280, D-6900 Heidelberg, GFR

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Institut für Zell- und Tumorbiologie, Deutsches Krebsforschungszentrum

Im Neuenheimer Feld 280, D-6900 Heidelberg, GFR

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,

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.

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,

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.

Search for other works by this author on:

Institut für Zell- und Tumorbiologie, Deutsches Krebsforschungszentrum

Im Neuenheimer Feld 280, D-6900 Heidelberg, GFR

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Hartmut Land, Manuel Grez, Hansjörg Hauser, Werner Lindenmaier, Günther Schütz, 5′-Terminal sequences of eucaryotic mRNA can be cloned with high efficiency, Nucleic Acids Research, Volume 9, Issue 10, 25 May 1981, Pages 2251–2266, https://doi.org/10.1093/nar/9.10.2251
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Abstract

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 × 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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