Cystic Fibrosis Genotyping by Direct PCR Analysis of Guthrle Blood Spots

In the United States the most common cystic fibrosis (CF) alleles known are F$08, G$$1D, G$42X, R$S3X, and N1303K. These mutations comprise 8 5 % of U.S. CF alleles, and their detection along with analysis of XV-2C and KM-19 restriction fragment length polymorphisms (RFLPs) can enable the determination of CF status. To facilitate studies for determining CF carrier status, we developed methods to dete(t each of these mutations and RFLPs by direct PCR amplification of dried blood spots collected on newborn screening (Guthrle) cards. Following collection, samples were protected from contamination by individual plastic bags. One-mm z segments of filter paper were added directly to 100-1~1 PCR reactions containing 1 /16 mM spermidine. Three Initial cycles at 96~ then $$~ for 3 min were performed to free DNA and minimize inhibition by other related materials. Next, 1 unit of Toq polymerase was added and a 2-min extension was carrled out at 72~ followed by 33 amplification cycles using denaturing, annealing, and extension temperatures and times optimal for each primer set. Then, 35 I~1 of each reaction was run on 8% acrylamide gels directly or 1% agarose gels following digestion; genotypes were Inferred by ethidlum bromide staining of gels. Guthrle 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 hapIotypes were determined. Our data Indicate: (1) direct PCR amplification provides a simple, rapid, and inexpensive method to determine CF genotypes directly from dried blood spots and (2) these methods provide special advantages regarding ease and convenience of sample collection, shipment, analysis, and storage