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Nucleic Acids Res ; 32(2): e18, 2004 Jan 23.
Article in English | MEDLINE | ID: mdl-14742865

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 (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.


Subject(s)
Biosensing Techniques/methods , Muscular Atrophy, Spinal/genetics , Polymorphism, Single Nucleotide/genetics , Biosensing Techniques/instrumentation , Cell Line , Cyclic AMP Response Element-Binding Protein , DNA/analysis , DNA/genetics , Fiber Optic Technology , Genotype , Kinetics , Muscular Atrophy, Spinal/pathology , Nerve Tissue Proteins/genetics , Oligonucleotide Probes/genetics , Polymerase Chain Reaction/methods , RNA-Binding Proteins , SMN Complex Proteins , Sensitivity and Specificity , Time Factors
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