Rapid cycle allele-specific amplification: Studies with the cystic fibrosis ΔF508 locus

Abstract

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 ΔF508 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 ΔF508 locus were amplified with the 3′ end of a discriminating primer at the ΔF508 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 °C or lower); with conventional cycling and "hot starts" (160-s cycles), only annealing temperatures of 61-62°C sufficiently discriminated between alleles. With rapid cycling, genotype could still be assessed with annealing temperatures as low as 25°C. We conclude that faster temperature cycling can improve the results of allele-specific amplification.