Sequencing Double-Stranded Linear DNA with Sequenase and [α-35S] d.ATP

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.