SBIR-STTR Award

DNA fingerprinting, or profiling, is widely used for paternity cases and criminal investigations (forensics). Current methodologies are cumbersome and often produce uninterpretable results. We have developed oligonucleotides containing a minor groove binder (MGB) that greatly enhance the performance and applicability of the 5' nuclease (fluorescence quench release or Taqman) assay in the discrimination of single nucleotide polymorphisms (SNPs). We propose to develop a kit for rapid DNA finger- printing using our proprietary MGB-conjugated oligonucleotide probes. A panel of 20 previously described diallelic polymorphisms comprise the markers of the fingerprint. The probability that two unrelated individuals would have matching fingerprints is less that 1 in 107. In this phase I effort, we will design and optimize the PCR primers and probes for the 20 loci, and demonstrate function in 5' nuclease assays on a set of known DNA samples. In phase II, we will increase the throughput of the 20-marker panel by multiplexing. PROPOSED COMMERCIAL APPLICATION: A genotyping (DNA fingerprinting, profiling,) kit for paternity testing and forensic applications. The same technology could be used for diagnosis of genetic diseases and also to categorize patients in clinical studies correlating genotypic markers with response to treatment.

DNA fingerprinting, or profiling, is widely used for paternity cases and criminal investigations (forensics). Current methodologies are cumbersome, expensive and often fail to produce interpretable results. A minor groove binder (MGB) has been developed that greatly enhances the ability of oligonucleotide probes to discriminate single nucleotide polymorphisms (SNPs) in the 5' nuclease (Taqman (registered), real time PCR, fluorescence quench release) assay. The goal is to develop a kit for rapid high throughput DNA fingerprinting using probes that contain MGB's and other proprietary modifications. The fingerprint is derived from a panel of 23 diallelic polymorphisms. The probability that two unrelated individuals would have matching fingerprints is approximately 1 in 10 (exp) 8. In the Phase I effort, PCR primers and probes for the 23 loci were optimized and their function was demonstrated in 5' nuclease assays using a set of known DNA samples. The goal of phase II is to develop a commercial prototype with increased throughput and reduced cost. Application of four spectrally separated reporter fluorophores and a novel quencher will enable multiplexed assays. Standard affordable instruments will replace expensive instruments specialized for 5' nuclease assays. The technologies developed herein can be readily applied to assays of any SNP's. Huge potential markets exist for these technologies in clinical diagnostics and pharmacogenomics. PROPOSED COMMERCIAL APPLICATION: The commercial prototype presented here is a kit for human identification in paternity testing. The technologies developed herein for single nucleotide polymorphism (SNP) screening could have wide market potentials in a variety of applications including clinical diagnostics and pharmacogenomics.