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Clinical Chemistry 53: 1372-1376, 2007. First published May 17, 2007; 10.1373/clinchem.2006.081810
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(Clinical Chemistry. 2007;53:1372-1376.)
© 2007 American Association for Clinical Chemistry, Inc.

Technical Briefs

Microtransponder-Based Multiplex Assay for Genotyping Cystic Fibrosis

Xin Lin1, James A. Flint1, Marco Azaro1, Thomas Coradetti1, Wesley M. Kopacka1, Deanna L. Streck2, Zhuying Wang1, James Dermody2 and Wlodek Mandecki1,a

1 PharmaSeq, Inc., Monmouth Junction, NJ;
2 Department of Microbiology & Molecular Genetics, University of Medicine and Dentistry, New Jersey-New Jersey Medical School, Newark, NJ;

aaddress correspondence to this author at: PharmaSeq, Inc., 11 Deer Park Drive, Suite 104, Monmouth Junction, NJ 08852; fax 732-355-0102, e-mail mandecki{at}pharmaseq.com


Abstract

Background: We developed and evaluated a genotyping assay for detection of 50 cystic fibrosis (CF) mutations. The assay is based on small (500 µm) electronic chips, radio frequency (RF) microtransponders (MTPs). The chips are analyzed on a unique fluorescence and RF readout instrument.

Methods: We divided the CF assay into 4 panels: core, Hispanic, African-American, and Caucasian. We amplified 18 CF transmembrane regulator (CFTR) DNA fragments covering 50 mutations by use of multiplex PCR using 18 CFTR gene-specific primer pairs. PCR was followed by multiplex allele-specific primer extension (ASPE) reactions and hybridization to capture probes synthesized on MTPs. We used 100 ASPE primers and 100 capture probes. We performed fluorescence measurements of hybridized MTP kits and assay analysis using a custom automated bench-top flow instrument.

Results: We validated the system by performing the assay on 23 commercial DNA samples in an internal study and 32 DNA samples in an external study. For internal and external studies, correct calls were 98.8% and 95.7%, false-positive calls 1.1% and 3.9%, and false-negative calls 0.12% and 0.36%, respectively.

Conclusions: The MTP-based multiplex assay and analysis platform can be used for CF genotyping.






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Copyright © 2007 by the American Association for Clinical Chemistry.