We are developing a novel microfluidic system that is composed of modular units that are fabricated from polymers, such as PMMA and PC, using a metal master to hot emboss microparts. Each unit possesses a discrete function and are interconnected using capillaries. The integrated system, in its final form, will perform the following functions, PCR amplification of the input DNA, purification of the PCR products, cycle sequencing using dye-terminator chemistry, purification of sequencing ladders using solid-phase reversible immobilization (SPRI) and electrophoresis sorting of the DNA products. All of these process steps can be completed in about 30 mins (16-channels). Fluorescence readout of the electrophoresis chip will be accomplished using a near-IR laser scanning microscope. This microscope not only collects steady-state emission, but also time resolved data. Using a two-color, four-lifetime approach, the fluorescence detector will possess the ability to process 8-spectroscopically unique fluorescent probes, which will permit processing two different templates simultaneously in the same gel tract. The thermal cycler will amplify DNA and perform cycle sequencing at a speed of 16 s/cycle and consume approximately 150 nL of reagents, minimizing sequencing cost by reducing reagent consumption. The purification chips will perform capillary electrochromatography for PCR product isolation and SPRI on PC surfaces activated by UV light to create surface carboxylate groups. The electrophoresis chip will contain an anionic mesoporous membrane to allow sample pre-concentration of the sequencing ladders to aid in detection. Labeling dyes will consist of appropriately prepared phthalocyanines (Pc) that contain water solubilizing groups that also alter the absorption properties of the base Pc to match the lasing wavelengths of the two-color microscope (680 and 780 nms). To change the fluorescence lifetimes, different metals will be incorporated into the core of the macrocycle. To evaluate the performance of the integrated system for DNA sequencing, we will look at gene conversion/replacement events between filled Alu chromosomal locations as well as the removal of these elements from the genome. PCR of 2,000 alleles for each locus from geographically diverse human genome samples will be undertaken and sequenced directly using the modular microfluidic system.
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