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.
Shen
Z
Z
Department of Radiological Health Sciences, Colorado State University, Fort Collins 80523.
Liu
J
J
Wells
R L
RL
Elkind
M M
MM
D000818:Animals; D019388:Cytochrome P-450 CYP1A2; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D051379:Mice; D010088:Oxidoreductases / Q000235:genetics; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D013696:Temperature; D013698:Templates, Genetic
D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019388:Cytochrome P-450 CYP1A2