Nucleic Acid Analysis via Hybridization-Induced Microbead Aggregation: Opportunities for Genetic Testing

Abstract

The association of genetic alterations with disease onset, progression, and sensitivity to therapeutics has opened exciting opportunities for the implementation of a personalized model of patient care. The practical translation of these findings into our mainstream medical regime requires nucleic acid analysis technology that is affordable, timeefficient, and simple to execute. In an effort to provide an attractive alternative to conventional methods, which are typically unsuitable for routine scenarios, this work builds on a unique, bead-based technique for sequence-specific DNA detection known as Hybridization-Induced Aggregation (HIA). HIA involves a pair of magnetic bead-bound oligonucleotide probes designed to hybridize to a complementary target sequence. Upon target-probe hybridization, the beads become tethered together, resulting in opticallydetectable bead aggregation.Initially, analytical instrumentation, including a "dual-force aggregation" platform as well as a microdevice for integrated PCR amplification and HIA detection, were engineered to maximize the utility of bead aggregation assays in terms of throughput, speed, and sensitivity. In subsequent work, the HIA technique was exploited for the detection of single nucleotide polymorphisms. This was demonstrated for the detection of KRAS mutations in lung and colorectal cancers in order to predict patient sensitivity to epidermal growth factor receptor-targeted therapies. Additionally, HIA detection was combined with multiplex allele-specific PCR to detect three important mutations [CYP2C9 *2, CYP2C9 *3, and VKORC1 (1173C>T)] that allow appropriate dosing of the ! iii common oral anticoagulant, warfarin. Overall, this work represents practical steps forward in the development of nucleic acid analysis technology that is amenable for routine clinical care, in order to ultimately reap the rewards of a personalized medical regime.