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Related Experiment Videos

Cellular gels. Purifying and mapping long DNA molecules.

P H Dear1, P R Cook

  • 1Sir William Dunn School of Pathology, Oxford, U.K.

The Biochemical Journal
|February 1, 1991
PubMed
Summary
This summary is machine-generated.

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Researchers developed a new

Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • Long DNA molecules (>0.1 Mbp) are fragile and easily sheared during standard isolation and manipulation.
  • Existing methods for analyzing large DNA fragments, such as pulsed-field gel electrophoresis, often result in DNA breakage upon recovery from gels.
  • This limits the purification and detailed analysis of large genomic fragments.

Purpose of the Study:

  • To develop a novel method for the recovery of intact, large DNA fragments from gels.
  • To enable facile manipulation and downstream applications of purified large DNA fragments.
  • To facilitate the restriction mapping of large DNA molecules, including chromosomes and yeast artificial chromosomes.

Main Methods:

  • Development of a novel 'cellular' gel matrix for DNA electrophoresis.

Related Experiment Videos

  • Embedding cells in agarose to protect chromosomal DNA.
  • Fractionation of embedded DNA using pulsed-field gel electrophoresis.
  • Recovery of resolved DNA fragments from the novel gel matrix without shear damage.

    • Main Results:

    • The novel 'cellular' gel matrix allows for the recovery of resolved DNA fragments (0.1-1.0 Mbp) in an intact state.
    • The recovered DNA fragments are amenable to facile manipulation without shear-induced breakage.
    • Demonstrated utility by successfully mapping baker's yeast chromosome III (approx. 0.36 Mbp).

    Conclusions:

    • The 'cellular' gel matrix provides a robust method for preserving the integrity of large DNA fragments during recovery.
    • This technique significantly advances the purification and restriction mapping of large DNA molecules.
    • The method is expected to be highly valuable for analyzing yeast artificial chromosomes and other large DNA constructs.