Cycle sequencing.

Stratagene Cloning Systems, La Jolla, California 92037 The PCR technique has revolutionized the way research is performed. PCR is being used in an ever increasing number of new techniques. One related technique that has emerged is cycle sequencing, or linear amplification sequencing, as it is sometimes known. (1'2) Cycle sequencing utilizes a thermostable DNA polymerase and dideoxynucleotide triphosphates to generate chain-termination sequence with a temperature-cycling format (Fig. 1). There are two basic differences between cycle sequencing and PCR amplification. The first is the presence of only one primer in the cycle-sequencing reaction used to prime synthesis of one strand of the DNA. The second difference is the presence of dideoxynucleotide triphosphates in the sequencing reactions that create the base-specific terminations required. The result of the temperature cycling is linear amplification of the sequencing product leading to an increase in the signal generated during the sequencing reaction when compared with standard sequencing protocols. Cycling the sequencing reactions results in several advantages: (1) The amount of template necessary for the sequencing reaction is greatly reduced; (2) because smaller amounts of template are added, fewer impurities are introduced, meaning less template preparation is required; and (3) the high temperature at which the sequencing reactions are run and the multiple heat-denaturation steps allow doublestranded templates such as plasmids, cosmids, X DNA, and PCR products to be sequenced reliably without a separate denaturation step.