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Continuous flow thermal cycler microchip for DNA cycle sequencing.

Hong Wang1, Jifeng Chen, Li Zhu

  • 1Department of Chemistry, Center for Bio-Modular Multi-Scale Systems, Louisiana State University, Baton Rouge, LA 70803, USA.

Analytical Chemistry
|September 2, 2006
PubMed
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A polymer-based continuous flow thermal cycler (CFTC) microchip enables rapid Sanger sequencing. This reusable microchip achieves long DNA read lengths efficiently, simplifying the sequencing workflow.

Area of Science:

  • Biotechnology
  • Molecular Biology
  • Analytical Chemistry

Background:

  • Sanger sequencing is a cornerstone of molecular biology.
  • Traditional thermal cyclers can be time-consuming and require significant hands-on manipulation.
  • Microfluidic devices offer potential for miniaturization and automation in molecular diagnostics.

Purpose of the Study:

  • To develop and evaluate a polymer-based continuous flow thermal cycler (CFTC) microchip for Sanger cycle sequencing.
  • To assess the performance of the CFTC chip in terms of read length, reaction time, and reusability.
  • To integrate the CFTC chip with a solid-phase reversible immobilization (SPRI) microchip for automated sample purification.

Main Methods:

  • A polycarbonate (PC) CFTC microchip with a 20-loop spiral channel was fabricated using hot embossing.

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  • The microchip featured three temperature zones (95°C, 55°C, 60°C) for denaturation, renaturation, and extension.
  • Sequencing cocktails were hydrodynamically pumped through the microreactor at varying linear velocities (1-12 mm/s).
  • The CFTC chip was coupled to a PC-based SPRI microchip for post-reaction purification.

    • Main Results:

    • A DNA read length of >600 bp was achieved with a 36-s extension time (4 mm/s linear velocity) in a total reaction time of 14.6 min.
    • Increased flow rates reduced reaction time but decreased read length.
    • The CFTC chip demonstrated high reusability (>30 runs) with minimal carryover contamination or performance degradation.
    • Coupling with the SPRI chip yielded read lengths comparable to benchtop instruments without manual manipulation.

    Conclusions:

    • The polymer-based CFTC microchip is an effective platform for rapid and efficient Sanger cycle sequencing.
    • The microchip's reusability and integration with SPRI purification streamline the DNA sequencing workflow.
    • This microfluidic approach offers a promising alternative to conventional methods for DNA sequencing applications.