Two-temperature PCR and heteroduplex detection: application to rapid cystic fibrosis screening.
We describe a rapid two-temperature PCR protocol for amplification of genomic DNA applied to the region of the most common mutation (delta F508) of the cystic fibrosis gene. Amplification products are detected as homo- or heteroduplexes on polyacrylamide gels as previously described. Data using two-temperature PCR show complete concordance with allele-specific hybridization after classical three-temperature PCR in 105 normal, carrier and affected individuals. Clinical application is demonstrated in a family which was uninformative by traditional RFLP linkage analysis. Two-temperature PCR may offer advantages of speed and specificity over three-temperature PCR in many clinical and research applications.
Dodson
L A
LA
Department of Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, Philadelphia.
Kant
J A
JA
D001483:Base Sequence; D003550:Cystic Fibrosis / Q000175:diagnosis / Q000235:genetics*; D004252:DNA Mutational Analysis; D015342:DNA Probes; D004591:Electrophoresis, Polyacrylamide Gel; D005784:Gene Amplification; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000032:analysis*; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D013696:Temperature
D015342:DNA Probes; D009692:Nucleic Acid Heteroduplexes; D009838:Oligodeoxyribonucleotides