An integrated multiple capillary array electrophoresis system for high-throughput DNA sequencing

Abstract

<p>Capillary array electrophoresis system was chosen to perform DNA sequencing because of several advantages such as rapid heat dissipation, multiplexing capabilities, gel matrix filling simplicity, and the mature nature of the associated manufacturing technologies;There are two major concerns for the multiple capillary systems. One concern is inter-capillary cross-talk and the other concern is excitation and detection efficiency. Cross-talk is eliminated through proper optical coupling, good focusing and immersing capillary array into index matching fluid. A side-entry excitation scheme with orthogonal detection was established for large capillary array. Two 100 capillary array formats were used for DNA sequencing. One format is cylindrical capillary with 150 [mu]m o.d., 75 [mu]m i.d and the other format is square capillary with 300 [mu]m out edge and 75 [mu]m inner edge. The capillary arrays were sandwiched between two fused silica plates with immersion solution in between, which formed a channel to guide the light. Different immersion fluids were chosen for different capillary formats. Immersion oil with a higher refractive index was chosen for the cylindrical capillary due to a higher trapping efficiency. Fused silica index matching liquid was chosen for the square capillary array because of the elimination of stray light produced during the laser beam transmission through the two dielectric media of capillary walls and immersion oil. The waveguide cell is designed in such a way so that the highest excitation efficiency is obtained. Comparing the two capillary formats, the square capillary array shows higher excitation efficiency and is scalable up to hundreds of capillaries which matches the requirement of high throughput for large scale DNA sequencing;This project is focused on the development of excitation and detection of DNA as well as performing DNA sequencing. The DNA injection schemes are discussed for the cases of single and bundled capillaries. An individual sampling device was designed. The base-calling was performed for a capillary from the capillary array with the accuracy of 98%.</p>