Rapid detection of single nucleotide polymorphisms associated with spinal muscular atrophy by use of a reusable fibre-optic biosensor |
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Authors: | Watterson James H Raha Sandeep Kotoris Christopher C Wust Christopher C Gharabaghi Farhad Jantzi Sarah C Haynes Nicole K Gendron Nathalie H Krull Ulrich J Mackenzie Alex E Piunno Paul A E |
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Institution: | Chemical Sensors Group, University of Toronto at Mississauga, 3359 Mississauga Road North, Mississauga, Ontario, Canada. |
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Abstract: | Rapid (<2 min) and quantitative genotyping for single nucleotide polymorphisms (SNPs) associated with spinal muscular atrophy was done using a reusable (approximately 80 cycles of application) fibre-optic biosensor over a clinically relevant range (0–4 gene copies). Sensors were functionalized with oligonucleotide probes immobilized at high density (~7 pmol/cm2) to impart enhanced selectivity for SNP discrimination and used in a total internal reflection fluorescence detection motif to detect 202 bp PCR amplicons from patient samples. Real-time detection may be done over a range of ionic strength conditions (0.1–1.0 M) without stringency rinsing to remove non-selectively bound materials and without loss of selectivity, permitting a means for facile sample preparation. By using the time-derivative of fluorescence intensity as the analytical parameter, linearity of response may be maintained while allowing for significant reductions in analysis time (10–100-fold), permitting for the completion of measurements in under 1 min. |
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