Selectivity of Hybridization Controlled by the Density of Solid Phase Synthesized DNA Probes on Glass Substrates
Optical biochip design based on varying the density of immobilized single-stranded DNA (ssDNA) oligonucleotide probes was examined. A method of immobilization was developed to yield various densities of probe molecules using photochemical activation of surfaces and in situ solid phase synthesis for DNA immobilization. High surface density of ssDNA probe (up to 1 × 1013 probes/cm2) was obtained using the immobilization method. The densities and extent of hybridization of nucleic acids were determined using confocal fluorescence microscopy. Selective hybridization of targets associated with spinal muscular atrophy containing single nucleotide polymorphisms (SNP), and their thermal denaturation profiles were investigated to examine the sensitivity and selectivity for SNP detection. The detection limit was less than 16 pM at room temperature. Single base mismatch discrimination was demonstrated based on control of melt temperature by selection of probe density, and temperature differences of 12-15°C could be achieved for SNP determination. Importantly, the results demonstrate that poor control of probe density can result in significant variability of selectivity, as seen by melt temperature shifts of up to 5°C in the density range that was investigated.
KeywordsNucleic acid hybridization fluorescence density single nucleotide polymorphism immobilization
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