Microsystem for Rapid DNA Sequencing

LSU Reference: 9613

Inventors:

Description:
  • DNA sequencing

Applications:
  • Genome Research
  • Genetic research
  • Biotechnology
  • Rapid medical or veterinary diagnostics

Advantages:
  • Speed
  • Uses much less reagent than standard technique, and is therefore less expensive
  • Suitable for highly multiplexed, automated DNA sequencing
  • Highly efficient

Abstract: A system is disclosed for the rapid and cost-effective sequencing of DNA. There are three principal components of the system: (1) a microreactor, which prepares DNA sequencing "ladders" using solid-phase techniques, preferably in capillary tubes whose volumes are on the order of 10-1000 nanoliters, preferably 10-200 nanoliters; (2) a microfabricated electrophoresis capillary separation unit, which separates the components of the sequencing ladders by size; and (3) a fluorescence detector with single-mode optical fibers interfaced directly to the electrophoresis capillary, for detecting and identifying the bases separated in the capillary. The system is suitable for a highly multiplexed, automated DNA sequencing device. Typical steps in sequencing are as follows: (1) PCR amplification of a DNA template in microtiter dishes using labelled primers, e.g., primers labelled with biotin; (2) immobilizing the labelled PCR products on the walls of one or more capillary tubes having volumes on the order of 10-200 nanoliters; (3) preparing nanoliter quantities of labelled Sanger extension products of the amplified DNA; (4) purifying the oligonucleotide sequencing ladders; (5) high speed electrophoretic separation of the sequencing ladders; and (6) near-infrared, laser-induced fluorescence detection of the oligonucleotides. Base-calling is preferably performed in a single lane format with a single fluorophore, in which the bases are distinguished by different fluorescence lifetimes of dyes that have otherwise have similar absorption and fluorescence emission spectra at the wavelengths used. Typical read lengths are on the order of 400-500 bases. Fluorescence is performed on- chip with one single-mode optical fiber carrying the excitation light to the capillary channel, and a second single-mode optical fiber collecting the fluorescent photons. Only sub-microliter volumes of expensive sequencing reagents and dye-labeled ddNTPs are required in this system. Compared to prior methods, there is a significant reduction in reagent costs -- by a factor of about 100 -- due to the substantial reduction in sample volumes.