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Related Experiment Videos

Versatile low-viscosity sieving matrices for nondenaturing DNA separations using capillary array electrophoresis

R S Madabhushi1, M Vainer, V Dolnik

  • 1Molecular Dynamics, Inc., Sunnyvale, CA 94086, USA.

Electrophoresis
|January 1, 1997
PubMed
Summary
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This study introduces a novel polyethyleneoxide (PEO) formulation for capillary electrophoresis (CE) that achieves high-resolution separation of double-stranded DNA (dsDNA) fragments from 80 bp to 40 kbp without pulsed fields.

Area of Science:

  • Molecular Biology
  • Analytical Chemistry
  • Biotechnology

Background:

  • High-resolution separation of double-stranded DNA (dsDNA) is crucial for physical mapping and PCR product analysis.
  • Conventional capillary electrophoresis (CE) requires pulsed fields for resolving DNA fragments larger than 23 kilobase pairs (kbp).

Purpose of the Study:

  • To develop a single formulation for broad-range dsDNA separation (80 bp-40 kbp) using CE without pulsed fields.
  • To optimize a sieving medium for enhanced DNA fragment resolution and detection limits.

Main Methods:

  • Utilized a low-viscosity sieving medium composed of a mixture of polyethyleneoxide (PEO) with different molecular weights (0.5% PEO (Mn 10(6)) and 0.1% PEO (Mn 8 x 10(6))).
  • Tested compatibility with intercalating dyes and capillary wall coatings.

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  • Optimized injection times (≤5 s) and field strengths (approx. 100 V/cm) for optimal resolution.

    • Main Results:

    • Achieved separation of dsDNA fragments from 80 bp to 40 kbp in a single run without pulsed fields.
    • Demonstrated a detection limit of 25 fg for a 200 bp DNA standard using Vistra Green.
    • Obtained complete sample runs within 70 minutes, enabling high-throughput analysis via capillary array electrophoresis (CAE).
    • Separated a size range approximately 10 times greater than conventional slab gel electrophoresis.

    Conclusions:

    • The developed PEO-based sieving medium offers a significant advancement in dsDNA separation by CE, expanding the accessible size range and eliminating the need for pulsed fields.
    • This method provides a robust, high-throughput solution for analyzing a wide spectrum of DNA fragments, applicable to various molecular biology and diagnostic applications.