Development and Application of Sequence Specific DNA Detection through Hybridization-induced Aggregation

The interrogation of genomic DNA for the presence of specific sequences is paramount for most biological assays, including clinical diagnostics, functional genomics, food safety and forensic DNA analysis. Here we present a new detection modality, based on hybridization of the target sequence to probes bound to paramagnetic particles. When hybridization occurs, the particles aggregate together in a visual manner, confirming the presence target DNA with a limit of detection of 100 fM for ssDNA. A systematic study into the effect sequence length alterations have on detection sensitivity is demonstrated. The specificity of HIA enables the detection of 1, 2 or 3 single point mutations in the target DNA, working towards applying HIA to future mutation detection. HIA was adapted for the detection of dsDNA, enabling the label-free detection of Salmonella enterica, bacteriophage for the diagnosis of multi-drug resistant tuberculosis and for the detection of TPOX for human identification. Furthermore, HIA is interfaced with PCR on a plastic integrated microdevice, where multiplexed IR-PCR is demonstrated for the first time. The microdevice combines low cost and minimal hardware to provide an easy to use system capable of PCR and HIA detection in 30 minutes. In addition, a direct-PCR protocol from whole blood was developed to aid in forensic investigations which was then adapted for use on the integrated device. Therefore, direct-PCR from whole blood and HIA detection can be performed in one hour. Overall, HIA is a new powerful modality in DNA detection technology appropriate for implementation at the point of care.