Rapid cycle allele-specific amplification: studies with the cystic fibrosis delta F508 locus.
Rapid cycle DNA amplification is a polymerase chain reaction technique with improved product specificity and cycle times of 20-60 s, allowing complete 30-cycle reactions in 10-30 min. The presence or absence of the delta F508 deletion and wild-type allele was determined in 104 cystic fibrosis patients by rapid cycle DNA amplification. In separate allele-specific assays, sequences on both sides of the delta F508 locus were amplified with the 3' end of a discriminating primer at the delta F508 locus, with either a 3-bp or a 1-bp mismatch. With rapid cycling (35-s cycles), single-base discrimination was achieved over a broad range of annealing temperatures (50 degrees C or lower); with conventional cycling and "hot starts" (160-s cycles), only annealing temperatures of 61-62 degrees C sufficiently discriminated between alleles. With rapid cycling, genotype could still be assessed with annealing temperatures as low as 25 degrees C. We conclude that faster temperature cycling can improve the results of allele-specific amplification.
Wittwer
C T
CT
Department of Pathology, University of Utah Medical School, Salt Lake City 84132.
Marshall
B C
BC
Reed
G H
GH
Cherry
J L
JL
D001483:Base Sequence; D003550:Cystic Fibrosis / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005838:Genotype; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D013696:Temperature; D013997:Time Factors
C505032:CFTR protein, human; D008565:Membrane Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator