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Related Experiment Videos

T7 DNA polymerase in automated dideoxy sequencing.

T Kristensen1, H Voss, C Schwager

  • 1European Molecular Biology Laboratory, Heidelberg, FRG.

Nucleic Acids Research
|April 25, 1988
PubMed
Summary

T7 DNA polymerase offers improved DNA sequencing accuracy over the Klenow fragment. Unmodified T7 polymerase successfully sequenced a DNA region unreadable by other enzymes, highlighting its utility in automated sequencing.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Automated DNA sequencing relies on efficient DNA polymerases for accurate nucleotide incorporation.
  • Variations in signal intensity during sequencing can lead to ambiguous base calls, particularly in repetitive regions.
  • The Klenow fragment of DNA polymerase I is a commonly used enzyme, but has limitations in signal uniformity.

Purpose of the Study:

  • To evaluate the performance of T7 DNA polymerase, both unmodified and chemically modified, in automated dideoxy DNA sequencing.
  • To compare the signal intensity uniformity and accuracy of T7 DNA polymerase against the Klenow fragment.
  • To identify specific sequencing challenges addressable by T7 DNA polymerase.

Main Methods:

  • Utilized T7 DNA polymerase (unmodified and 3'-5' exo-nuclease deficient) and Klenow fragment for dideoxy sequencing.

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  • Employed an automated sequencing system with fluorescence-labeled primers and real-time detection.
  • Analyzed signal intensity variations, focusing on the 'C' track and preceding nucleotide effects.

    • Main Results:

    • T7 DNA polymerase exhibited more uniform signal intensities compared to the Klenow fragment.
    • Low 'C' signal intensities were often preceded by a 'T', an effect modulated by neighboring nucleotides.
    • Sequences ambiguous with the Klenow enzyme were resolved using T7 polymerase.
    • Unmodified T7 polymerase uniquely resolved one sequence unreadable by Klenow fragment or modified T7 polymerase.

    Conclusions:

    • T7 DNA polymerase enhances the accuracy and reliability of automated DNA sequencing.
    • The enzyme's performance, particularly the unmodified form, is advantageous for resolving difficult sequences.
    • T7 DNA polymerase represents a valuable tool for improving DNA sequencing workflows.