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5′-Terminal sequences of eucaryotic mRNA can be cloned with high efficiency
Open accessAbstract
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.
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Cited by
A simple and very efficient method for generating cDNA libraries
Gubler, U., & Hoffman, B. J. (1983) · Gene · doi:10.1016/0378-1119(83)90230-5 · Cited by 4,510
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Yamamoto, T., Davis, C. G., Brown, M. S., Schneider, W. J., Casey, M. L., Goldstein, J. L., & Russell, D. W. (1984) · Cell · doi:10.1016/0092-8674(84)90188-0 · Cited by 1,382
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Basler, K., Oesch, B., Scott, M., Westaway, D., Wälchli, M., Groth, D. F., McKinley, M. P., Prusiner, S. B., & Weissmann, C. (1986) · Cell · doi:10.1016/0092-8674(86)90662-8 · Cited by 835
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Metadata
- Authors
- Land, H., Grez, M., Hauser, H., Lindenmaier, W., & Schütz, G.
- Venue
- Nucleic Acids Research
- Publisher
- Oxford University Press (OUP)
- Published
- 1981
- Volume
- 9
- Issue
- 10
- Pages
- 2251-2266
- ISSN
- 0305-1048 (ISSN-L)
- 1362-4962
- OpenAlex
- W2039816194
- Cited by
- 361
- References
- 0
- FWCI
- 25.55
- Type
- journal article
- Language
- en
- Open access
- green
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- Land, H., Grez, M., Hauser, H., Lindenmaier, W., & Schütz, G. (1981). 5′-Terminal sequences of eucaryotic mRNA can be cloned with high efficiency. Nucleic Acids Research. https://doi.org/10.1093/nar/9.10.2251
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