Cycle sequencing using degenerate primers containing inosines.

A modified protocol for cycle sequencing DNA amplified by polymerase chain reaction (PCR) is described. The method involves two sequential linear PCR amplifications using a small amount of double-stranded DNA as a template and a stringent annealing temperature: 1) alpha-35S-dATP labeling of specific primers initially in degenerate primer mixture and 2) dideoxy-ribonucleotide termination of the extended and alpha-35S-dATP-labeled specific primers. The method does not require end labeling and is useful in sequencing PCR-amplified DNA sequences from highly degenerate primers when the sequences of the regions flanking those to be primed are unknown.