Rapid Isothermal Amplification and Multiplexed Surface Detection of Short DNA Sequences
E. Tan, Y. Han, E. Kniazeva, M. Buechel, B. Erwin and A. Niemz
Keck Graduate Institute, US
DNA isothermal amplification, DNA nanospheres, DNA functionalized nanoparticles, surface based detection, DNA microarrays, atomic force microscopy, impedance detection, electronic DNA detection
We have coupled a novel isothermal amplification method for short oligonucleotides (EXPAR) with solution and surface-based detection involving DNA-functionalized gold nanospheres. In these assays, a trigger oligonucleotide is exponentially amplified and converted to a reporter oligonucleotide. Detection is based on DNA-functionalized gold nanospheres, which are either aggregated in solution or immobilized on a surface. The assay allows for different target sequences to be detected using the same set of DNA:Au nanospheres through appropriately designed reporter sequences. The solution based assay relies on simple visual detection, mitigated by the shift in plasmon resonance upon DNA nanosphere aggregation, which results in a visually detectable color change. The overall assay is rapid, sensitive and sequence-specific, with minimal requirement for instrumentation. We have implemented a multiplexed surface based variation of this assay. To investigate the sensitivity and specificity of this multiplexed format, we have performed fluorescence based detection on standard DNA microarrays, or atomic force microscopy (AFM) of DNA nanosphere immobilization on silicon substrates. Our ultimate goal is to utilize this assay for the sensitive, specific, multiplexed, impedance based electronic DNA detection on an array of semiconductor electrodes.
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Nanotech 2007 Conference Program Abstract