Direct sequencing of double-stranded PCR products with the sequenase kit and [alpha-35S] dATP.

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 (), which we describe in detail below.