BACKGROUND: We developed and evaluated a genotyping assay for detection of 50 cystic fibrosis (CF) mutations. The assay is based on small (500 microm) 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.
BACKGROUND: We developed and evaluated a genotyping assay for detection of 50 cystic fibrosis (CF) mutations. The assay is based on small (500 microm) 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.
Authors: I Gryczynski; E G Matveeva; P Sarkar; S Bharill; J Borejdo; W Mandecki; I Akopova; Z Gryczynski Journal: Chem Phys Lett Date: 2008-10 Impact factor: 2.328
Authors: Gregory S Richmond; Htet Khine; Tina T Zhou; Daniel E Ryan; Tony Brand; Mary T McBride; Kevin Killeen Journal: PLoS One Date: 2011-04-22 Impact factor: 3.240