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Improvement in sensitivity of allele-specific PCR facilitates reliable noninvasive prenatal detection of cystic fibrosis
Journal article   Open access

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

Ourania Nasis, Shanel Thompson, Tom Hong, Margaret Sherwood, Shawn Radcliffe, Laird Jackson, Tomas Otevrel and Sara W Thompson
Clinical chemistry (Baltimore, Md.), v 50(4), pp 694-701
Apr 2004
DOI: https://doi.org/10.1373/clinchem.2003.025981
PMID: 14764639
Featured in Collection :   UN Sustainable Development Goals @ Drexel
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https://doi.org/10.1373/clinchem.2003.025981View
Published, Version of Record (VoR)Maybe Open Access (Publisher Bronze),  Open

Abstract

Adult Alleles Cystic Fibrosis - blood Cystic Fibrosis - diagnosis Cystic Fibrosis - genetics Cystic Fibrosis Transmembrane Conductance Regulator - blood Cystic Fibrosis Transmembrane Conductance Regulator - genetics Female Gestational Age Humans Male Mutation Polymerase Chain Reaction - methods Pregnancy Prenatal Diagnosis Sensitivity and Specificity
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. 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. 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. 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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