Abstract
The Polymerase Chain Reaction (PCR) products are doublestranded linear DNA molecules. Although hybridization may provide some information on the amplified products, clearcut identification of nucleic acids is best achieved by sequencing. When PCR fragments are heterogeneous, cloning in vectors is compulsory for sequencing. In some cases, however, PCR products are 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), that is described in detail below.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Casanova, J. L, Pannetier, C., Jaulin, C., and Kourilsky, P. (1990)Optimal conditions for directly sequencing double-stranded PCR products with sequenase. Nucl. Acids Res 18, 4028.
Musette, P, Casanova, J.-L., Labbaye, C., Dorner, M., Kourilsky, P, and Cayre, Y (1991) Wegener’s autoantigen and leukemia Blood 77, 1398–1399.
Casanova, J.-L, Romero, P., Widmann, C., Kourilsky, P., and Maryanski, J.L. (1991) T cell receptor genes m a series of class I Major Histocompatibility Complex restricted cytotoxic T lymphocyte clones spectfic for a Pfasmodwm berghei nonapeptide: tmplications for T cell allelic exclusion and anttgen-specificrepertoire. J, Exp. Med. 174, 1371–1383.
Jager, R. J., Anvret, M, Hall, K., and Scherer, G (1990) A human XY female with a frame shaft mutation in the candidate testis-determming gene SRY. Nature (London) 348, 452–454.
Nakayama, K.-I., Tokito, S., Pannetier, C, Nakauchi, H., and Gachelm, G. (1991) MHC gene Q8/gd of the BALB/cJ mouse stram cannot encode a Qa-2,3 class I antigen.Immunogenetics 33, 225–234.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1993 Humana Press Inc. Totowa, New Jersey
About this protocol
Cite this protocol
Casanova, JL. (1993). Sequencing Double-Stranded Linear DNA with Sequenase and [α-35S] d.ATP. In: Griffin, H.G., Griffin, A.M. (eds) DNA Sequencing Protocols. Methods in Molecular Biology™, vol 23. Humana Press. https://doi.org/10.1385/0-89603-248-5:191
Download citation
DOI: https://doi.org/10.1385/0-89603-248-5:191
Publisher Name: Humana Press
Print ISBN: 978-0-89603-248-4
Online ISBN: 978-1-59259-510-5
eBook Packages: Springer Protocols