Cystic Fibrosis Diagnosis by Genetic Bit Analysis

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