11] Specific primer-directed DNA sequence analysis using automated fluorescence detection and labeled primers

Abstract

This chapter presents the current chemistries involved in the synthesis of dye-primer sets for use with the Applied Biosystems 373A automated DNA sequencer as well as reliable protocols for their utilization in enzymatic DNA sequencing. It discusses some selection rules for assessing candidate primer sequences. There are three primary applications of specific primers: (1) the sequencing of nucleic acids flanked by the same primer sequence, such as cloning vector-specific (so-called “universal”) primers or primers for the routine repetitive sequence analysis of poly allelic genetic regions, such as in the determination of human leukocyte antigen (HLA) haplotypes, (2) the filling of occasional small gaps in extended sequence obtained by random sequencing methods, and (3) classic specific primer-directed sequencing (SPDS), often termed gene walking, wherein the entire sequence of a large DNA is obtained by walking along its length using information obtained from a previous round of sequence analysis to select a new primer for a subsequent round.