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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)

Abstract

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.

Keywords

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.

References

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Copyright information

© Humana Press Inc., Totowa, NJ 1996

Authors and Affiliations

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

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