A forensic analysis system will be developed based on analysis of non-pseudoautosomal regions of the Y chromosome and the hypervariable region of the mitochondrial genome. The intent is to increase the probability of discrimination for DNA evidence collected in sexual assault cases and from mass disaster sites. The methodology will be based on the detection of single nucleotide polymorphisms (SNP) by genetic bit mapping (GBA) techniques that are already used by Molecular Tool (a recent acquisition of GeneScreen Inc). The proposed technology for identifying SNPs has been designed for high throughput analysis. It involves the following analytical steps 1. PCR of region of interest using a regular and a phosphorothioated primer. 2. Preparation of phosphorothioated ssDNA using a T7 exonuclease (unprotected strand is digested). 3. Capture of ssDNA to immobilized primers (two plates in parallel for detection of all four bases). 4. Single base extension of the primer with a dideoxynucleoside triphosphate labeled with biotin or fluorescein. 5. Detection of the extension product via the biotin or fluorescein tag using alkaline phosphatase-labeled anti- fluorescein or peroxidase labeled antibiotin. Bound alkaline phosphatase is detected first and then the well is washed and any bound peroxidase is measured (two color detection). Initially, the method will be developed on a macroscale using 96-well microtiter plates and colorimetric detection. Subsequently, it will be miniaturized onto glass slides and employ direct fluorescent detection of captured enzyme labeled conjugate, ultimately using four different fluorescent labels so that all four nucleotides at each SNP locus can be interrogated at once.
