Cystic fibrosis genotyping by direct PCR analysis of Guthrie blood spots

In the United States the most common cystic fibrosis (CF) alleles known are F508, G551D, G542X, R553X, and N1303K. These mutations comprise approximately 85% of U.S. CF alleles, and their detection along with analysis of XV-2C and KM-19 restriction fragment length polymorphisms (RFLPs) can enable the determination of CF status. To facilitate studies for determining CF carrier status, we developed methods to detect each of these mutations and RFLPs by direct PCR amplification of dried blood spots collected on newborn screening (Guthrie) cards. Following collection, samples were protected from contamination by individual plastic bags. One-mm2 segments of filter paper were added directly to 100-microliters PCR reactions containing 1/16 mM spermidine. Three initial cycles at 96 degrees C, then 55 degrees C, for 3 min were performed to free DNA and minimize inhibition by other related materials. Next, 1 unit of Taq polymerase was added and a 2-min extension was carried out at 72 degrees C, followed by 33 amplification cycles using denaturing, annealing, and extension temperatures and times optimal for each primer set. Then, 35 microliters of each reaction was run on 8% acrylamide gels directly or 1% agarose gels following digestion; genotypes were inferred by ethidium bromide staining of gels. Guthrie blood spots of 250 CF probands and their parents were screened and the frequencies of all five mutations as well as the XV-2C KM-19 RFLP haplotypes were determined.(ABSTRACT TRUNCATED AT 250 WORDS)