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) α-35S-dATP labeling of specific primers initially in degenerative primer mixture and 2) dideoxyribonucleotide termination of the extended and α-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.
|Original language||English (US)|
|State||Published - Jan 1 1993|
All Science Journal Classification (ASJC) codes
- Biochemistry, Genetics and Molecular Biology(all)