Rapid detection of single nucleotide polymorphisms associated with spinal muscular atrophy by use of a reusable fibre-optic biosensor.
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 (approximately 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.
Watterson
James H
JH
Chemical Sensors Group, University of Toronto at Mississauga, 3359 Mississauga Road North, Mississauga, Ontario, Canada.
Raha
Sandeep
S
Kotoris
Christopher C
CC
Wust
Christopher C
CC
Gharabaghi
Farhad
F
Jantzi
Sarah C
SC
Haynes
Nicole K
NK
Gendron
Nathalie H
NH
Krull
Ulrich J
UJ
Mackenzie
Alex E
AE
Piunno
Paul A E
PA
D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D002460:Cell Line; D017362:Cyclic AMP Response Element-Binding Protein; D004247:DNA / Q000032:analysis / Q000235:genetics; D005336:Fiber Optic Technology; D005838:Genotype; D007700:Kinetics; D009134:Muscular Atrophy, Spinal / Q000235:genetics* / Q000473:pathology; D009419:Nerve Tissue Proteins / Q000235:genetics; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D016601:RNA-Binding Proteins; D055532:SMN Complex Proteins; D012680:Sensitivity and Specificity; D013997:Time Factors
D017362:Cyclic AMP Response Element-Binding Protein; D009419:Nerve Tissue Proteins; D015345:Oligonucleotide Probes; D016601:RNA-Binding Proteins; D055532:SMN Complex Proteins; D004247:DNA