Tools for Ultraspecific Probe/Primer Design

摘要

We offer a novel methodology for rapidly identifying superior- performance DNA probes/primers for use in detecting emerging or engineered pathogens. Our approach will deliver DNA probes and PCR primers that have an unprecedentedly low probability of false positives or confusion by environmental background, and which resist evasion by threat agent engineering. Any detection method that utilizes DNA or RNA probes or primers will benefit greatly by using probes/primers designed with our methodologies. This technology is made possible by novel insights into statistical properties of useful probes, primer pairs, and targets. Such findings have become possible because of dramatic advances in the computational analysis of genomic sequence data. Using our novel approach, background sequences are rigorously (not heuristically, e.g., BLAST) discriminated against. Thus, probes and primers developed using these tools can be known to be at least three mismatches away from the nearest other sequence in an entire set of DNA sequences employed in the calculations. The Phase I studies will demonstrate the advantages of our design technology. In this phase we will (1) perform extensive analysis of several Category A and B pathogens and produce(deliver) the database of all human and/or 'background' 1, 2, 3, and 4 mismatches blind 16-22-mers present in their genomes; (2) transform in house scientific software into a Windows-based application that allows users to perform similar calculations for any custom sequence for 16-19-mers with up to 3 mismatches blind; and (3) perform intensive experimental validation in order to verify candidate sequences and experimentally estimate false discovery rate.