The objective of this grant is to demonstrate that Molecular Tool's solid-phase method of direct DNA mutation identification, called Genetic Bit Analysis (GBA), can significantly expand the range of mutations analyzed and improve the power, capacity and cost-effectiveness of diagnosing the genotypically complex human disease cystic fibrosis. Molecular Tool's commercial goal is to develop flexibly formatted solid- phase GBA arrays that can simultaneously identify diagnostic, prognostic and therapeutic applications for a large number of CF mutations. The biochemical steps in GBA are as follows: 1) amplification of CF DNA by PCR using modified, exonuclease-resistant primers, 2) generation of a single-stranded template by exonuclease digestion, 3) hybridization of amplified templates to primers immobilized in microtiter plate wells, 4) incorporation of biotinylated dideoxynucleosides at the site of the mutant allele by DNA polymerase, 5) and enzymatic detection of the incorporated bases. Phase I of this grant aims to: 1) develop GBA reagents for 10 common CF mutations, 2) test these reagents on human genomic DNAs, 3) demonstrate the biochemical accuracy and reproducibly of GBA by testing 200 coded CF patient DNA samples 3) develop a protype test format. These data will allow accurate analysis of the Phase II development costs and operational features of a range of commercial CF test prototypes.
