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Related Experiment Videos

Multiple-complete-digest restriction fragment mapping: generating sequence-ready maps for large-scale DNA sequencing

G K Wong1, J Yu, E C Thayer

  • 1The Human Genome Center, Department of Medicine, University of Washington, Seattle, WA 98195, USA.

Proceedings of the National Academy of Sciences of the United States of America
|May 13, 1997
PubMed
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Multiple-complete-digest mapping creates detailed DNA maps by ordering clones and fragments. This technique accurately validates clones and sequence assemblies for large genomic regions.

Area of Science:

  • Genomics
  • Molecular Biology
  • Bioinformatics

Background:

  • DNA mapping is crucial for genome sequencing and analysis.
  • Existing methods may lack the resolution or redundancy needed for complex genomic regions.
  • High-resolution mapping facilitates accurate clone selection for sequencing.

Purpose of the Study:

  • To demonstrate the practicality of multiple-complete-digest mapping for large and complex genomic targets.
  • To assess the accuracy of maps generated by this technique for validating sequencing data.
  • To establish a robust method for creating cosmid-based maps of human genomic regions.

Main Methods:

  • Utilized multiple-complete-digest mapping with three enzymes on yeast artificial chromosome contigs.
  • Subcloned yeast artificial chromosomes into cosmids at high redundancy (x15-30).

Related Experiment Videos

  • Generated DNA fingerprints, electrophoresed fragments, and used fluorescent scanning for imaging.

    • Main Results:

    • Successfully produced cosmid-based maps exceeding one million base pairs for human genome regions 7q31.3 and 7p14.
    • Ordered nearly all restriction fragments, enabling the selection of minimal tiling paths with small clone overlaps.
    • Developed a process that rejects aberrant clones, ensuring map accuracy.

    Conclusions:

    • Multiple-complete-digest mapping is practical and accurate for large-scale genomic mapping.
    • The generated maps are sufficiently accurate for validating selected sequencing clones and shotgun sequence assemblies.
    • This technique offers a reliable approach for advancing genomic research and sequencing projects.