Direct Sequencing of Polymerase Chain Reaction Products

The advantages of direct sequencing of polymerase chain reaction (PCR) products over conventional sequencing of cloned, single-stranded DNA are manifold. Speed is perhaps the greatest asset. Time-consuming creation and screening of libraries, fragment purification, subcloning, bacterial transfection, and plasmid preparation steps are all eliminated. Cost is an advantage for similar reasons. The ability to sequence thousands or even millions of different templates at once, thereby obtaining pooled averages of mutated or polymorphic sequences, is another benefit. Moreover, short DNA sequences not obtainable by conventional cloning, such as DNA from paraffin-embedded tissues, can be sequenced using PCR sequencing.