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PMID: 6166921 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

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

Nucleic acids research ·Vol. 9 ·No. 10 ·1981-05-25 ·Pages 2251-66

Land H, Grez M, Hauser H, Lindenmaier W, Schütz G

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 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.

MeSH Terms
Avian Myeloblastosis Virus/enzymology Base Sequence Cloning, Molecular DNA, Recombinant/metabolism Muramidase/genetics Plasmids RNA, Messenger/genetics RNA-Directed DNA Polymerase
Chemicals
DNA, Recombinant RNA, Messenger RNA-Directed DNA Polymerase Muramidase
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Land H
Grez M
Hauser H
Lindenmaier W
Schütz G
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32 references, click to expand
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/9.10.2251
Published
1981-05-25
Pages
2251-66
Language
English
Region
England
NLM ID
0411011
PMCID
PMC326843
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/9.10.2251 PMC Full Text View journal details
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