Direct Sequencing of Double-Stranded PCR Products with the Sequenase Kit and [α-35S] dATP
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.
KeywordsVortex Urea EDTA Agarose Bromide
- 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.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.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.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