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Cloning specific complete polyadenylylated 3'-terminal cDNA segments.

K M Lang, R A Spritz

    Gene
    |January 1, 1985
    PubMed
    Summary

    This study presents a novel method for extending partial complementary DNA (cDNA) clones, ensuring complete 3' termini and poly(A) tails. This technique facilitates the isolation of specific gene segments, aiding in synthetic mRNA production and genetic variation discovery.

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    Area of Science:

    • Molecular Biology
    • Genetics
    • Biotechnology

    Background:

    • Partial complementary DNA (cDNA) clones are valuable tools in molecular biology.
    • Extending these clones to obtain complete 3' ends and polyadenylated sequences is often necessary for downstream applications.
    • Existing methods may have limitations in efficiency or specificity.

    Purpose of the Study:

    • To describe a simple and effective method for extending partial cDNA clones.
    • To enable the isolation of specific 3' cDNA segments, including poly(A) tails.
    • To generate templates for in vitro transcription of synthetic mRNAs.

    Main Methods:

    • The method combines plasmid-primed first-strand cDNA synthesis with second-strand synthesis by primer extension.
    • This approach specifically selects for double-stranded cDNAs with complete 3' termini.
    • The technique was applied to extend existing cDNA clones of human beta- and G gamma-globin.

    Main Results:

    • Successful extension of partial cDNA clones was achieved, yielding products with complete 3' termini and poly(A) tails.
    • Specific 3' segments of human beta- and G gamma-globin cDNAs were isolated.
    • Nucleotide sequence analysis revealed a previously unreported sequence polymorphism in the 3' untranslated region of the gamma-globin cDNA.

    Conclusions:

    • The described method provides a straightforward way to generate full-length 3' cDNA sequences.
    • The isolated cDNA recombinants are useful for constructing templates for synthetic polyadenylylated mRNA and pre-mRNA production.
    • The identification of sequence polymorphisms contributes to understanding genetic variation.

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