Heterozygote and mutation detection by direct automated fluorescent DNA sequencing using a mutant Taq DNA polymerase.
We describe a method for direct cycle sequencing of PCR fragments amplified from genomic DNA or cDNA. DNA sequencing template is amplified using PCR and oligonucleotide primers flanking the region of interest. The amplified fragment is directly cycle sequenced using fluorescent sequencing primers, Sanger dideoxy sequencing chemistry and an enzyme mixture of a mutant Taq DNA polymerase and thermostable pyrophosphatase. The sequence ladders produced are analyzed on a real-time, automated four-color sequencing system. The method produces sequence ladders from unpurified PCR fragments of sufficiently high quality such that heterozygotes can be reproducibly detected and identified by software that recognizes signal-strength patterns indicative of mixed-base positions.
Chadwick
R B
RB
Perkin-Elmer Corporation, Applied Biosystems Division, Foster City, CA, USA.
Conrad
M P
MP
McGinnis
M D
MD
Johnston-Dow
L
L
Spurgeon
S L
SL
Kronick
M N
MN
D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D005091:Exons / Q000235:genetics; D005456:Fluorescent Dyes; D006580:Genetic Carrier Screening / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*; D019914:Taq Polymerase
D017931:DNA Primers; D005456:Fluorescent Dyes; D019914:Taq Polymerase; D012324:RNA-Dependent RNA Polymerase; D004259:DNA-Directed DNA Polymerase