Micro-electrophoresis devices with integrated fluorescence detectors and reactors for high-throughput DNA sequencing

S. A. Soper, S. Ford, J. Davies, D. Williams, B. Cheng, M. Klopf, G. Calderon, V. Saile

Research output: Contribution to journalConference article

1 Scopus citations

Abstract

This work describes the development of micro-devices for high-throughput DNA sequencing applications. Basically, two research efforts will be discussed; (1) fabrication and characterization of micro-reactors to prepare Sanger chain terminated DNA sequencing fragments on a nanoliter scale and; (2) X-ray photolithography of PMMA substrates for the high aspect ratio preparation of electrophoresis devices. The micro-reactor consisted of a 5'-biotinylated catfish olfactory gene (1 Kbp), which was amplified by PCR, and attached to the interior wall of an aminoalkylsilane derivatized fused-silica capillary tube (volume = 31-196 nL) via a streptavidin/biotin linkage. Coverage of the interior capillary wall with biotinylated DNA averaged 77% (± 10%). Stability of the anchored template under pressure and electroosmotic rinsing was favorable, requiting ∼150 h of continuous rinsing to reduce the coverage by only 50%. The capillary micro-reactor was placed inside an air thermocycler to control temperature during Sanger ddNTP chain extension and directly coupled to a capillary separation column filled with an LPA solution via low dead volume capillary interlocks. The complimentary DNA fragments generated in the reactor were heat denatured from the immobilized template and directly injected onto a gel-filled capillary using electropumping for size fractionation and detection using NIR-LIF analysis. The total amount of termination fragments in the 31 nL reactor volume was estimated to be 5.2×10-13 moles and sequencing was shown to produce read lengths on the order of 400 bases. Work will also be described concerning the development of micro-electrophoresis devices in X-ray sensitive photoresists using LIGA techniques. An electrophoresis device with an integrated fluorescence detector was constructed for the high resolution separation of DNA oligonucleotides. The choice of substrate for the electrophoresis was PMMA, due to its intrinsic low electroosmotic flow. Using X-ray lithography in PMMA substrates, the aspect ratios associated with the micromachining was estimated to be >10,000:1.

Original languageEnglish (US)
Pages (from-to)36-46
Number of pages11
JournalProceedings of SPIE - The International Society for Optical Engineering
Volume2985
DOIs
StatePublished - Dec 1 1997
EventUltrasensitive Biochemical Diagnostics II - San Jose, CA, United States
Duration: Feb 10 1997Feb 12 1997

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Condensed Matter Physics
  • Computer Science Applications
  • Applied Mathematics
  • Electrical and Electronic Engineering

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