Truncated fragments in polymerase chain reaction-based DNA sequencing

Reconstitution experiments were performed by using an ordinary dye-primer p rotocol of a template spiked with known amounts of truncated fragments. We observed that as little as 0.2 mole-percentage of the truncated fragment ca used sequence interpretation problems. Two protocols were developed for seq uencing with dye-labeled terminators; this eliminates the problems with tru ncated fragments, which are adapted to a one-dye chemistry. One was designe d for single extension sequencing using T7 DNA polymerase and one for cycle sequencing. To avoid precipitation and centrifugation and to facilitate au tomation, the dye-terminator protocols included the use of a biotinylated s equencing primer. Thus, the Sanger fragments were recovered and, by magneti c separation, washed and released by formamide, EDTA, and heat treatment be fore loading on the electrophoresis gel. Integrated procedures for sequenci ng PCR products using one-dye-labeled terminators suitable for automation a re described. High quality data in terms of long reads and detection of pol ymorphisms is obtained. The protocols serve as attractive alternatives to i nternal labeling and dye-primer approaches.