Proviral DNA Cloning

  • Julie Overbaugh
  • Steven Dewhurst
  • James I. Mullins


There are two primary considerations when preparing to clone a retrovirus, particularly HIV. The first is choosing the source of viral DNA, and the second is selecting an appropriate cloning vector.


Polymerase Chain Reaction Current Protocol Polymerase Chain Reaction Protocol Host Bacterium Infectious Molecular Clone 
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  1. 1.
    Shaw, G. M., Hahn, B. H., Arya, S. K., Groopman, J. E., Gallo, R. C., and Wong-Staal, F. 1984. Molecular characterization of human T-cell leukemia virus type III in the Acquired Immune Deficiency Syndrome. Science 226:1165–1171.PubMedCrossRefGoogle Scholar
  2. 2.
    Harper, M. H., Marselle, L. M., Gallo, R. C., and Wong-Staal, F. 1986. Detection of lymphocytes expressing human T-lymphotropic virus type III in lymph nodes and peripheral blood from infected individuals by in situ hybridization. Proc Natl Acad Sci USA 83:772–776.PubMedPubMedCentralCrossRefGoogle Scholar
  3. 3.
    Psallidopoulos, M. C., Schnittman, S. M., Thompson III, L. M., Baseler, M., Fuci, A. S., Lane, H. C., and Salzman, N. P. 1989. Integrated proviral human immunodeficiency virus type 1 is present in CD4+ peripheral blood lymphocytes in healthy seropositive individuals. J Virol 63:4626–4631.PubMedPubMedCentralGoogle Scholar
  4. 4.
    Simmonds, P., Balfe, P., Peutherer, J. F., Ludlam, C. A., Bishop, J. O., and Brown, A. J. L. 1990. Human immunodeficiency virus-infected individuals contain provirus in small numbers of peripheral mononuclear cells and at low copy numbers. J Virol 64:864–872.PubMedPubMedCentralGoogle Scholar
  5. 5.
    Overbaugh, J., Donahue, P. R., Quackenbush, S. L., Hoover, E. A., and Mullins, J. I. 1988. Molecular cloning of a feline leukemia virus that causes fatal immunodeficiency disease in cats. Science 239:906–910.PubMedCrossRefGoogle Scholar
  6. 6.
    Meyerhans, A., Cheynier, R., Albert, J., Selh, M., Kwok, S., Sninsky, J., Morfeldt-Manson, L., Asjo, B., and Wain-Hobson, S. 1989. Temporal fluctuations in HIV quasispecies in vivo and not reflected by sequential HIV isolations. Cell 58:901–910.PubMedCrossRefGoogle Scholar
  7. 7.
    Asjo, B., Morfeldt-Manson, L., Albert, J., Biberfeld, G., Karlsson, A., Lidman, K. E., and Fenyo, E. M. 1986. Replicative capacity of human immunodeficiency virus from patients with varying severity of HIV infection. Lancet 2:660–662.PubMedGoogle Scholar
  8. 8.
    Kwok, S. 1990. Procedures to minimize PCR-product carry-over. In PCR Protocols: A Guide to Methods and Applications, M. A. Innis, D. H. Gelfand, J. J. Sninsky and T. J. White, eds., Academic Press, New York, pp. 142–145.Google Scholar
  9. 9.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  10. 10.
    Ausubel, F.M. 1987. Current Protocols in Molecular Biology, F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, eds., Wiley and Sons, New York.Google Scholar
  11. 11.
    Hirt, B. 1967. Selective extraction of polyoma DNA from infected mouse cell culture. J Mol Biol 26:365–369.PubMedCrossRefGoogle Scholar
  12. 12.
    Kornfeld, H., Riedel, N., Viglianti, G., Hirsch, V., and Mullins, J. I. 1987. Cloning of HTLV-IV and its relation to simian and human immunodeficiency viruses. Nature 326:610–613.PubMedCrossRefGoogle Scholar
  13. 13.
    Alizon, M., Sonigo, P., Barre-Sinoussi, F., Chermann, J.-C., Tiollais, P., Montagnier, L., and Wain-Hobson, S. 1984. Molecular cloning of lymphadenopathy-associated virus. Nature 326:757–760.CrossRefGoogle Scholar
  14. 14.
    Luciw, P. A., Potter, S. J., Steimer, K., Dina, D., and Levy, J. A. 1984. Molecular cloning of AIDS-associated retrovirus. Nature 312:760–763.PubMedCrossRefGoogle Scholar
  15. 15.
    Fischaud, A.-M., Lehrach, H., Poustka, A., and Murray, N. 1983. Lambda replacement vectors carrying polylinker sequences. J Mol Biol 170:827–842.CrossRefGoogle Scholar
  16. 16.
    Dunn, I. S., and Blattner, F. R. 1987. Charon 36 to 40: Multi enzyme, high capacity, recombination deficient replacement vectors with polylinkers and polystuffers. Nucleic Acids Res 15:2677–2698.PubMedPubMedCentralCrossRefGoogle Scholar
  17. 17.
    Short, J. M., Fenandez, J. M., Sorge, J. A., and Huse, W. D. 1988. ZAP: A bacteriophage expression vector with in vivo excision properties. Nucleic Acids Res 16:7583–7600.PubMedPubMedCentralCrossRefGoogle Scholar
  18. 18.
    Maniatis, T., Fritsch, E. F., and Sambrook, J. 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  19. 19.
    Dewhurst, S., Embretson, J. E., Fultz, P. N., and Mullins, J. I. 1990. PCR amplification, molecular cloning and sequence analysis of SIVSMMPBj14. Submitted for publication.Google Scholar
  20. 20.
    Kaiser, K., and Murray, N.E. 1985. The use of lambda replacement vectors and construction of representative genomic DNA libraries. In DNA Cloning, A Practical Approach, D. M. Glover, ed., IRL Press, Oxford, England, pp. 1–47.Google Scholar
  21. 21.
    Yamamoto, K. R., Alberts, R., Benzinger, R., Lawhorne, L., and Trieber, G. 1970. Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large scale virus purification. Virology 40:734–745.PubMedCrossRefGoogle Scholar
  22. 22.
    Maniatis, T., Hardison, R. C., Lacy, E., Laurer, J., O’Connell, C., Quon, D., Sim, G. K., and Efstratiadis, A. 1978. The isolation of structural genes from libraries of eucaryotic DNA. Cell 15:687–701.PubMedCrossRefGoogle Scholar
  23. 23.
    Seed, B., Parker, R. C., and Davidson, N. 1982. Representation of DNA sequences in recombinant DNA libraries prepared by restriction enzyme partial digestion. Gene 19:201–209.PubMedCrossRefGoogle Scholar
  24. 24.
    Kusumi, K., Conway, B., Hirsch, M., Gallo, M. V., Cunningham, S., and Mullins, J. I. Unpublished observations.Google Scholar
  25. 25.
    Pääbo, S., Higuchi, R. G., and Wilson, A. C. 1989. Ancient DNA and the polymerase chain reaction. J Biol Chem 264:9709–9712.PubMedGoogle Scholar
  26. 26.
    Dower, W. J., Miller, J. F., and Ragsdale, C. W. 1988. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res 16:6127–6145.PubMedPubMedCentralCrossRefGoogle Scholar
  27. 27.
    Adachi, A., Gendelman, H. E., Koenig, S., Folks, T., Willey, R., Rabson, A., and Martin, M.A. 1986. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol 59:284–291.PubMedPubMedCentralGoogle Scholar
  28. 28.
    Schubert, M., Brayton, P. R., and Meier, E. 1988. Fusion of DNA sequences without the use of shared restriction sites. FOCUS (BRL) 10:8–9.Google Scholar
  29. 29.
    O’Connor, M., Peifer, M., and Bender, W. 1989. Construction of large DNA segments in Escherichia coli. Science 244:1307–1312.PubMedGoogle Scholar
  30. 30.
    Innis, M. A., and Gelfand, D. H. 1990. Optimization of PCRs. In PCR Protocols: A Guide to Methods and Applications, M. A. Innis, D. H. Gelfand, J. J. Sninsky and T. J. White, eds., Academic Press, New York, pp. 3–12.Google Scholar
  31. 31.
    Mullis, K. B., and Faloona, F. A. 1987. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 155:335–350.PubMedCrossRefGoogle Scholar
  32. 32.
    Faloona, F., and Mullis, K., personal communication.Google Scholar

Copyright information

© Stockton Press 1990

Authors and Affiliations

  • Julie Overbaugh
    • 1
  • Steven Dewhurst
    • 2
  • James I. Mullins
    • 3
  1. 1.Department of MicrobiologyUniversity of WashingtonSeattleUSA
  2. 2.Department of Cancer BiologyHarvard School of Public HealthBostonUSA
  3. 3.Department of Microbiology and ImmunologyStanford University School of MedicineStanfordUSA

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