Direct Sequencing of Double-Stranded PCR Products with the Sequenase Kit and [α-35S] dATP

  • Jean-Laurent Casanova
Part of the Methods in Molecular Biology™ book series (MIMB, volume 65)


The polymerase chain reaction (PCR) products are double-stranded linear DNA molecules. Although digestion with a set of restriction enzymes, hybridization with an internal probe, detection of a single-stranded chain polymorphism, or of a site for specific chemical cleavage may all provide useful information on the amplified products, clear-cut identification of nucleic acids is best achieved by sequencing. When a PCR fragment is heterogeneous, cloning in a vector may be required for sequencing each individual molecule independently. In many cases, however, regions of or often the entire PCR product is homogeneous, and direct sequencing without cloning may be undertaken. We have developed a simple and fast method for directly sequencing linear double-stranded DNA molecules, such as PCR products (1), which we describe in detail below.


Polymerase Chain Reaction Product Polymerase Chain Reaction Fragment Polyacrylamide Solution Asymmetrical Polymerase Chain Reaction Thermostable Polymerase 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Casanova, J.-L., Pannetier, C., Jaulin, C., and Kourilsky, P. (1990) Optimal conditions for directly sequencing double-stranded PCR products with sequenase. Nucleic Acids Res. 18, 4028.PubMedCrossRefGoogle Scholar
  2. 2.
    Casanova, J.-L., Romero, P., Widmann, C., Kourilsky, P., and Maryanski, J. L. (1991) T cell receptor genes in a series of class I Major Histocompatibility Complex restricted cytotoxic T lymphocyte clones specific for a Plasmodium berghei nonapeptide: implications for T cell allelic exclusion and antigen-specific repertoire. J. Exp. Med. 174, 1371–1383.PubMedCrossRefGoogle Scholar
  3. 3.
    Casanova, J.-L., Cerottini, J.-C., Matthes, M., Necker, A., Gournier, H., Barra, C., Widmann, C., MacDonald, H. R., Lemonmer, F., Malissen, B., and Maryanski, J. L. (1992) H-2 restricted cytolytic T lymphocytes specific for HLA display T cell receptors of limited diversity. J. Exp. Med. 176, 439–447.PubMedCrossRefGoogle Scholar
  4. 4.
    Casanova, J.-L., Martinon, F., Gournier, H., Barra, C., Regnault, A., Pannetier, C., Kourilsky, P., Cerottini, J.-C., and Maryanski, J. L. (1993) T cell receptor selection by and recognition of two class I MHC restricted antigenic peptides that differ at a single position. J. Exp. Med. 177, 811–820.PubMedCrossRefGoogle Scholar
  5. 5.
    Romero, P., Casanova, J.-L., Cerottini, J.-C., Maryanski, J. L., and Luescher, I. (1993) Differential TCR photoaffinity labeling among H-2Kd restricted CTL clones specific for a photoreactive peptide derivative. Labeling of the α chain correlates with Jα segment usage. J. Exp. Med. 177, 1247–1256.PubMedCrossRefGoogle Scholar
  6. 6.
    Maryanski, J. L., Jongeneel, C.-V., Bucher, P., Casanova, J.-L., and Walker, P. R (1996) Single-cell PCR analysis of TCR repertoire selected by antigen in vivo: a high magnitude CD8 response is comprised of very few clones. Immunity 4, 47–55.PubMedCrossRefGoogle Scholar
  7. 7.
    MacDonald, H. R., Casanova, J.-L., Maryanski, J. L., and Cerottini, J.-C. (1993) Oligoclonal expansion of MHC class I restricted CTL during a primary response in vivo: direct monitoring by flow cytometry and polymerase chain reaction. J. Exp. Med. 177, 1487–1492.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1996

Authors and Affiliations

  • Jean-Laurent Casanova
    • 1
  1. 1.Developpement Normal et PathologiqueINSERM U132, Hopital NeckerParisFrance

Personalised recommendations