A microarray based STR genotyping system utilizing RecA-mediated ligation and nan

DESCRIPTION (provided by applicant): Tandem repeat sequences (STR) are distributed widely in the human genome. These sites exhibit high levels of length polymorphism, as a result of their relative instability during DNA replication, i.e., the tendency for slippage to occur between the template and newly synthesized strands causing deletion or addition of repeat units. Changes in STR repeat number can lead to heritable genetic predispositions and disease. However, their variability makes them good markers fo r genetic studies. One class, the microsatellites containing 4 base pair repeat units, has been selected by the FBI as the basis for a genetic identification system, termed CODIS. Current analysis systems require a 1-2 day assay and employ electrophoretic separation of DNA fragments that contain the STR locus for accurate sizing and identification of the repeat number. The long-term goal of this project is to develop a simple, cost-effective and automatable assay system for STR analysis that allows human identification in less than 2 hours from sample to result by avoiding the need for electrophoretic separation and potentially avoiding the need for PCR amplification. In Phase I of this proposal we will use a single locus (TPOX) to demonstrate that a ccurate microarray based STR analysis is technically feasible and sufficiently rapid. In Phase II we will expand this assay to include several other CODIS loci, and attempt to increase assay speed further by eliminating the PCR step. Our approach t o achieving these goals combines the technologies of two companies. Gene Check, Inc. has developed RML technology in which RecA catalyzes specific base pairing of oligonucleotide probes at the STR locus with subsequent ligation into specific products that reflect the length of the STR locus. Each product is coded with a unique recognition sequence, which allows their separation by hybridization to a microarray. Nanosphere has developed an automated microarray-based assay platform with sufficient specificity and sensitivity to allow SNP detection from total genomic DNA without PCR amplification by employing nanoparticle detection technology. Our preliminary data suggest that the Phase I tasks can be accomplished within a 6-12 month period. This will l ay the groundwork for the proposed Phase II extension of this technology to several additional FBI CODIS STR loci, and assay and system designs to avoid PCR. Moreover, the results should allow us to assess the potential of this technology for medical appli cations, such as tissue typing, transplantation monitoring, and analysis of genetic conditions or predispositions mediated by relatively short nucleotide repeat polymorphisms.