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The development of a novel vector for construction of a full-length cDNA library

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Abstract

A new original vector pEM-(dT)40(f+) has been prepared. It can be used for cDNA library construction from polyadenylated mRNA, isolated from various sources. The vector pGEM-(dT)40f(+) is initially transformed into single stranded and then into a linear form and its (dT)40 tail at the 3′-end is used as the vector-primer for synthesis of the first strand cDNA. The use of a synthetic oligonucleotide complementary to the vector and recombinant DNA results in vector circularization and synthesis of the second strand cDNA. This approach has the following advantages: (1) it significantly simplifies cDNA library construction, which includes three steps; (2) full-length cDNA library construction is achieved by adding a (dC)n homopolymer tail to the 5′end; (3) preparation of a clone library requires a few milligrams of total RNA; (4) it is possible to obtain cDNA clones up to 10 kbp; (5) it does not require PCR reaction (which can induce artifact mutations in cDNA sequences); (6) this approach does not employ restrictase treatment and chimeric cDNA products are not formed.

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Authors and Affiliations

  1. Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, ul. Pogodinskaya 10, Moscow, 119121, Russia

    V. I. Fedchenko, A. A. Kaloshin & A. E. Medvedev

Authors
  1. V. I. Fedchenko
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  2. A. A. Kaloshin
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  3. A. E. Medvedev
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Correspondence to V. I. Fedchenko.

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Original Russian Text © V.I. Fedchenko, A.A. Kaloshin, A.E. Medvedev, 2010, published in Biomeditsinskaya Khimiya.

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Fedchenko, V.I., Kaloshin, A.A. & Medvedev, A.E. The development of a novel vector for construction of a full-length cDNA library. Biochem. Moscow Suppl. Ser. B 4, 353–361 (2010). https://doi.org/10.1134/S1990750810040050

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  • DOI: https://doi.org/10.1134/S1990750810040050

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