RAPID CYCLE ALLELE-SPECIFIC AMPLIFICATION - STUDIES WITH THE CYSTIC-FIBROSIS DELTA-F(508) LOCUS

Rapid cycle DNA amplification is a polymerase chain reaction technique with improved product specificity and cycle times of 20-60 s, allowin g complete 30-cycle reactions in 10-30 min. The presence or absence of the DELTAF508 deletion and wild-type allele was determined in 104 cys tic fibrosis patients by rapid cycle DNA amplification. In separate al lele-specific assays, sequences on both sides of the DELTAF508 locus w ere amplified with the 3' end of a discriminating primer at the DELTAF 508 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 ran ge of annealing temperatures (50-degrees-C or lower); with conventiona l cycling and ''hot starts'' (1 60-s cycles), only annealing temperatu res of 61-62-degrees-C sufficiently discriminated between alleles. Wit h rapid cycling, genotype could still be assessed with annealing tempe ratures as low as 25-degrees-C. We conclude that faster temperature cy cling can improve the results of allele-specific amplification.