Received on August 14, 2003
Accepted on January 13, 2004

Molecular Diagnostics and Genetics

Improvement in Sensitivity of Allele-specific PCR Facilitates Reliable Noninvasive Prenatal Detection of Cystic Fibrosis

¹ New Jersey Medical School, University of Medicine & Dentistry of New Jersey, Newark, NJ
² Center for Molecular Diagnostics and Genome Research, Department of Obstetrics and Gynecology, Drexel University College of Medicine, Philadelphia, PA

^* To whom correspondence should be addressed. E-mail: to34{at}drexel.edu.

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 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.