Rapid Isothermal Amplification and Multiplexed Surface-Based Detection of Short DNA Sequences

We have coupled a novel isothermal amplification method for short oligonucleotides (EXPAR) with solution and surface-based detection involving DNA-functionalized gold nanospheres. 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 through aggregation of DNA-Au nanospheres in solution, and is rapid, sensitive and sequence-specific, with minimal requirement for instrumentation. We have also implemented a multiplexed surface based variation of this assay using either 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 impedance based electronic DNA detection.