Sequencing of Mammalian Messenger RNA Using Oligonucleotide Primers: Application to Hypoxanthine Phosphoribosyltransferase and a Phosphoglycerate Kinase Variant

  • R. Joshi
  • F. F. Snyder
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 253A)


We have investigated the suitability of applying primer directed dideoxynucleotide mediated sequencing methods to the characterization of low abundance messages. Direct mRNA sequence analysis is based on the priming of a target within a heterogeneous population of RNA by using oligonucleotide primers complimentary to the mRNA of interest. We have examined the minimal message abundance limits and mRNA amounts required for direct dideoxynucleotide RNA sequencing as a means of exploring the utility of this approach in characterizing point mutations.


Phosphoglycerate Kinase Hypoxanthine Phosphoribosyltransferase Amino Acid Composition Analysis Mouse Neuroblastoma Cell Line Nonspherocytic Hemolytic Anemia 
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  1. 1.
    D.J. Jolly, H. Okayama, P. Berg, A.C. Esty, D. Filpula, P. Bohlen, G.G. Johnson, J.E. Shively, T. Hunkapillar, and T. Friedmann, Isolation and characterization of a full-length expressible cDNA for human hypoxanthine phosphoribosyl-transferase, Proc. Natl. Acad. Sci. U.S.A. 80: 477 (1983).PubMedCrossRefGoogle Scholar
  2. 2.
    A.M. Michelson, A.F. Markham, and S.H. Orkin, Isolation and DNA sequence of a full length cDNA clone for human X-chromosome-encoded phosphoglycerate kinase, Proc. Natl. Acad. Sci. U.S.A. 80: 472 (1983).PubMedCrossRefGoogle Scholar
  3. 3.
    P.H. Hamylyn, M.J. Gait, and C. Milstein, Complete sequence of an immunoglobulin mRNA using specific priming and dideoxynucleotide method of RNA sequencing, Nucleic Acids Res. 15: 1067 (1981).Google Scholar
  4. 4.
    J. Geliebter, R.A. Zeff, R.W. Melvold, and S.G. Nathenson, Mitotic recombination in germ cells generated two major histocompatibility complex mutant genes shown to be identical by RNA sequence analysis: K (bm9) and K (bm6), Proc. Natl. Acad. Sci. U.S.A. 83: 3371 (1986).PubMedCrossRefGoogle Scholar
  5. 5.
    J. Singer-Sam, R.L. Simmer, D.H. Keith, L. Shively, M. Teplitz, K. Itakura, S.M. Gartler, and A.D. Riggs, Isolation of a cDNA clone for human X-linked 3-phosphoglycerate kinase by use of a mixture of synthetic oligodeoxyribonucleotides as a detection probe, Proc. Natl. Acad. Sci. U.S.A. 80: 802 (1983).PubMedCrossRefGoogle Scholar
  6. 6.
    D.W. Melton, D.S. Konecki, D.H. Ledbetter, J.F. Hejmanik, and C.T. Caskey, In vitro translation of hypoxanthine-guanine phosphoribosyltransferase mRNA: characterization of a mouse neuroblastoma cell line that has elevated levels of hypoxanthine-guanine phosphoribosyltransferase protein, Proc. Natl. Acad. Sci. U.S.A. 78: 6977 (1981).PubMedCrossRefGoogle Scholar
  7. 7.
    R. Joshi and F.F. Snyder, in preparation.Google Scholar
  8. 8.
    H. Fujiia, S.-H. Chen, J. Akatsuka, S. Miwa, and A. Yoshida, Use of cultured lymphoblastoid cells for the study of abnormal enzymes: molecular abnormality of a phosphoglycerate kinase variant associated with hemolytic anemia, Proc. Natl. Acad. Sci. U.S.A. 78: 2587 (1981).CrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1989

Authors and Affiliations

  • R. Joshi
    • 1
  • F. F. Snyder
    • 1
  1. 1.Departments of Pediatrics and Medical Biochemistry Faculty of MedicineUniversity of CalgaryCalgaryCanada

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