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Efficient low redundancy large-scale DNA sequencing at EMBL

H Voss1, C Schwager, S Wiemann

  • 1Biochemical Instrumentation Programme, European Molecular Biology Laboratory, Heidelberg, Germany.

Journal of Biotechnology
|July 31, 1995
PubMed
Summary
This summary is machine-generated.

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A new DNA sequencing strategy using primer walking achieves high accuracy with low redundancy, minimizing sequencing reactions and overlap. This directed approach is more efficient than random shotgun methods for large-scale projects.

Area of Science:

  • Genomics
  • Molecular Biology
  • Bioinformatics

Background:

  • Cosmid-scale DNA sequencing requires efficient strategies for high-accuracy consensus sequence determination.
  • Random shotgun sequencing is a common but often redundant approach for large DNA fragments.

Purpose of the Study:

  • To develop and evaluate a directed DNA sequencing strategy with low redundancy for cosmid-scale projects.
  • To compare the efficiency and redundancy of primer walking against random sequencing strategies.

Main Methods:

  • Automated double-stranded Sanger dideoxy sequencing using inexpensive walking primers.
  • Utilized T7 DNA polymerase and internal labeling with fluorescein-15-dATP on A.L.F. DNA sequencers.
  • Employed 60-cm glass plates for extended read lengths up to 1000 bases.

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Main Results:

  • The directed primer walking strategy achieved the lowest redundancy (2.6–2.8) compared to random methods.
  • This strategy was successfully applied to cosmids from chromosomes IX, XI, and XV in the yeast genome project.
  • High-accuracy raw data were obtained with cost-effective reagents.

Conclusions:

  • Primer walking is a highly efficient, low-redundancy DNA sequencing strategy for cosmid-scale projects.
  • Future developments include a 2-dye sequencing device and an initial random phase to further reduce primer usage.
  • The optimized strategy maintains low redundancy while improving efficiency.