Improvement in sensitivity of allele-specific PCR facilitates reliable noninvasive prenatal detection of cystic fibrosis.

BACKGROUND Cell-free fetal DNA circulating in maternal blood has potential as a safer alternative to invasive methods of prenatal testing for paternally inherited genetic alterations, such as cystic fibrosis (CF) mutations. METHODS We used allele-specific PCR to detect mutated CF D1152H DNA in the presence of an excess of the corresponding wild-type sequence. Pfx buffer (Invitrogen) containing replication accessory proteins and Taq polymerase with no proofreading activity was combined with TaqMaster PCR Enhancer (Eppendorf) to suppress nonspecific amplification of the wild-type allele. The procedure was tested on DNA isolated from plasma drawn from 11 pregnant women (gestational age, 11-19.2 weeks), with mutation confirmation by chorionic villus sampling. RESULTS The method detected 5 copies of the CF D1152H mutant allele in the presence of up to approximately 100,000 copies of wild-type allele without interference from the wild-type sequence. The D1152H mutation was correctly identified in one positive sample; the only false-positive result was seen in a mishandled sample. CONCLUSIONS This procedure allows for reliable detection of the paternally inherited D1152H mutation and has potential application for detection of other mutations, which may help reduce the need for invasive testing.