Heterozygote and mutation detection by direct automated fluorescent DNA sequencing using a mutant Taq DNA polymerase.
Biotechniques
; 20(4): 676-83, 1996 Apr.
Article
em En
| MEDLINE
| ID: mdl-8800689
ABSTRACT
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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
RNA Polimerase Dependente de RNA
/
Análise Mutacional de DNA
/
Análise de Sequência de DNA
/
DNA Polimerase Dirigida por DNA
/
Triagem de Portadores Genéticos
Tipo de estudo:
Diagnostic_studies
Limite:
Humans
Idioma:
En
Revista:
Biotechniques
Ano de publicação:
1996
Tipo de documento:
Article