Phase I demonstrated the feasibility of developing a silicon microtransponder for use in three-dimensional arrays using nucleic acid probes. During Phase II we will prepare synthetic oligonucleotide probes complementary to viral gene nucleic acid sequences. We will: 1) identify one or more fluorophores suitable for the transponder-based assay; 2) develop and optimize derivitization methods for conjugating oligonucleotide probes to transponder surfaces, (including methods to derivatize glass supports), apply/modify these methods to derivitize the transponder's surface either chemically or by coating with organic substances or biopolymers and define optimum conditions for conjugating probes to the microtransponder's surface; 3) PCR-amplify gene target segments, incorporate fluorophores into these products, then hybridize amplified test nucleic acid oligomers to probes immobilized on the transponder; 4) determine the effect of target DNA length on hybridization efficiency; 5) optimize conditions for hybridization of target nucleic acids to the probes conjugated on the microtransponder surface, and; 6) determine assay point mutation discrimination by PCR amplification of gene target segments having one mutation, followed by fluorescent tag- labeling of targets and hybridization of target sequences to probes immobilized on the transponder. Three-dimensional microtransponder assays could prove superior to two-dimensional array assays and significantly reduce new probe development and assay test time.
Results of the proposed work can be applied in nucleic acid-based diagnostics in vitro for infectious diseases, genetic testing, forensic testing, fertility testing or in vitro fertilization. The results can be instrumental in development of a commercial assay for the HIV protease gene, which can aid the physician to choose the proper therapeutic regimen.
