The project will examine the potential of producing an extremely sensitive DNA measurement system based upon the transformation of the nucleotides in DNA to mononucleotide triphosphates followed by detection of the triphosphates using Luciferase. Two different strategies will be explored for the formation of nucleotide triphosphates from DNA: direct generation of the triphosphates by reversing the polymerization reaction of polymerases, and; digestion of the polymer with DNAses followed by transformation of the nucleotide monophosphates to triphosphates using coupled enzyme systems. Phase I of the work will be used to: a) determine the ability of various enzymes to carry out transformations of nucleotides at very low levels of substrate; b) purify in-house the required enzymes necessary for Phase II and; c) determine the detection limit for the combined system. The best systems will then be optimized during Phase II to achieve a detection limit below 100pgrams of DNA and the effect of many agents used to stabilize and purify protein will be determined. During Phase Iii, an automated system to perform the assay will be developed and software for automated data acquisition will be created.
The primary market for a DNA Quantitation Kit is the biological pharmaceutical industry. There is a strong F.D.A. mandated need to control the level of DNA contamination to a level less than 100pg. The success of this proposed research will yield one of the most sensitive methods for detecting trace levels of DNA contamination.
| Reed, Jennifer L; Vo, Thuy D; Schilling, Christophe H et al. (2003) An expanded genome-scale model of Escherichia coli K-12 (iJR904 GSM/GPR). Genome Biol 4:R54 |
