GENOTYPING WITH MINOR-GROOVE-BINDER-CONTAINING PROBES.

APPLICANT'S DESCRIPTION: 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.