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Related Experiment Videos

Probe mapping to facilitate transposon-based DNA sequencing.

L D Strausbaugh1, M T Bourke, M T Sommer

  • 1Department of Molecular and Cell Biology, University of Connecticut, Storrs 06269-3125.

Proceedings of the National Academy of Sciences of the United States of America
|August 1, 1990
PubMed
Summary
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Transposons like gamma delta (Tn1000) offer mobile primer sites for DNA sequencing. A new probe-mapping method efficiently locates and orients these sites, enabling large-scale sequencing without subcloning.

Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • DNA sequencing strategies require efficient methods for primer binding site localization.
  • Transposons offer mobile primer sites, but their location and orientation must be easily determined for optimal use.
  • Random distribution of insertion sites is crucial for efficient large-scale sequencing.

Purpose of the Study:

  • To develop and demonstrate an efficient probe-based method for localizing and orienting transposon-borne primer sites.
  • To assess the suitability of the gamma delta (Tn1000) transposon for DNA sequencing applications.
  • To evaluate the distribution of gamma delta insertion sites in cloned DNA fragments.

Main Methods:

  • Development of a probe-based method for transposon localization and orientation, independent of restriction mapping or sequence knowledge.

Related Experiment Videos

  • Application of the method to map and orient 49 gamma delta (Tn1000) insertions in a cloned Drosophila melanogaster DNA fragment.
  • Utilizing oligonucleotide primers specific to gamma delta segments for dideoxynucleotide sequencing.

    • Main Results:

    • The probe-based method efficiently determined the location and orientation of transposon insertions.
    • Gamma delta insertions were found to be randomly distributed across the DNA fragment, even in regions with varying base composition.
    • Insertions preferentially occurred in A+T-rich regions within G+C-rich DNA segments.
    • Successful sequencing of large DNA segments was achieved without the need for subcloning.

    Conclusions:

    • The developed probe-mapping technique is adaptable for large-scale DNA sequencing strategies.
    • The gamma delta (Tn1000) transposon is well-suited for providing mobile primer binding sites in DNA sequencing.
    • Transposon-based probe mapping allows for efficient sequencing of large cloned DNA segments, bypassing traditional subcloning methods.