The introduction of novel fluorescent dyes has significantly impacted DNA sequencing technologies by improving accuracy and read lengths, increasing throughput, and decreasing costs. Despite these advances, however, sequencing reactions labeled by dye-terminator chemistries show lower, although acceptable phred quality values. This results from sequence-dependent bias incorporation by DNA polymerase resulting in non-uniform peak heights in the chromatographic data. Variation in peak heights is particularly problematic when attempting to quantitate different base mixtures for heterozygote analyses of single nucleotide polymorphisms (SNPs). Bias incorporation can be attributed to steric hindrance from the large fluorescent dye structures attached to DNA bases via 3-amino-l-propynyl (AP-3) linkers, Here, we propose the development of fluorobases, which are modified DNA bases that fluoresce. We anticipate that the elimination of the AP-3 linkers and the compactness of the proposed fluorobase nucleotides will significantly reduce bias incorporation by DNA polymerase and therefore, significantly improve data quality and heterozygote identification. To test this concept, a prototypical series of novel fluorobase dye structures will be synthesized and their physical and spectral properties will be fully characterized. It is anticipated that these compounds will be highly soluble in aqueous solutions due to their polar structures. Although the development of the dyes proposed in this research are a priori intended for DNA sequencing applications, the commercial potential of these novel compounds is extremely high for general fluorescent labeling and detection of nucleic acids. Specifically, the scope of research outlines the synthesis of two different fluorobases, each consisting of either a purine or pyrimidine base. Following each successful synthesis and structure analysis, the fluorobases will be characterized spectroscopically to determine their excitation and emission maxima and respective half-bandwidths, and their molar extinction coefficients, quantum yields, photostability and solubility in a variety of aqueous and organic solvents. Subsequently, fluorobases, which show good fluorescent and physical properties, will be coupled to a 2',3'- dideoxyribose molecule and phosphorylated to create fluorobase nucleoside triphosphates. Oligo-template incorporation and conventional sequencing assays will be performed to examine DNA polymerase performance and bias incorporation effects. Successful outcome of this Phase I research could lead to the development of a set of spectrally resolvable fluorobase nucleotides configured as single dye or energy-transfer dye cassettes. The work described here could potentially impact the field of DNA sequencing technologies by increasing accuracy and read lengths, reducing costs, and improving heterozygote detection.
Thesaurus Terms: DNA, fluorescent dye /probe, nucleic acid sequence, technology /technique development nucleobase, purine, pyrimidine high performance liquid chromatography, mass spectrometry, nuclear magnetic resonance spectroscopy
