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Clinical Chemistry 50: 694-701, 2004. First published February 5, 2004; 10.1373/clinchem.2003.025981
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Right arrow Molecular Diagnostics and Genetics
(Clinical Chemistry. 2004;50:694-701.)
© 2004 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

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

Ourania Nasis1,1, Shanel Thompson1, Tom Hong1, Margaret Sherwood1, Shawn Radcliffe1, Laird Jackson1 and Tomas Otevrel1,a

1 Center for Molecular Diagnostics and Genome Research, Department of Obstetrics and Gynecology, Drexel University College of Medicine, Philadelphia, PA.

aAddress correspondence to this author at: Department of Obstetrics and Gynecology, Drexel University College of Medicine, 245 North 15th St., Mail Stop 495, Philadelphia, PA 19102. Fax 215-762-4090; 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.




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