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Related Experiment Videos

DNA sequence analysis with a modified bacteriophage T7 DNA polymerase.

S Tabor, C C Richardson

    Proceedings of the National Academy of Sciences of the United States of America
    |July 1, 1987
    PubMed
    Summary

    Chemically modified T7 DNA polymerase offers enhanced processivity and efficiency for DNA sequencing. This modification eliminates exonuclease activity and improves nucleotide analog incorporation, enabling accurate sequence determination.

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    Enzymatic labeling of DNA.

    Current protocols in human genetics·2008

    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Genetics

    Background:

    • DNA sequencing is crucial for genetic research and diagnostics.
    • Chain-termination method (Sanger sequencing) is a widely used technique.
    • Limitations of native DNA polymerases hinder sequencing accuracy and efficiency.

    Purpose of the Study:

    • To develop and characterize a modified T7 DNA polymerase for improved chain-termination DNA sequencing.
    • To enhance enzyme processivity, eliminate unwanted exonuclease activity, and improve nucleotide analog incorporation.

    Main Methods:

    • Chemical modification of phage T7 DNA polymerase.
    • Assessing enzyme processivity through nucleotide incorporation assays.
    • Evaluating 3' to 5' exonuclease activity.

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  • Testing efficiency with nucleotide analogs and dITP substitution for dGTP.

    • Main Results:

    • The modified T7 DNA polymerase exhibits high processivity, synthesizing thousands of nucleotides.
    • Elimination of 3' to 5' exonuclease activity prevents degradation of synthesized DNA.
    • Efficient incorporation of nucleotide analogs leads to uniform radioactive intensity of terminated fragments.
    • Substitution of dITP for dGTP eliminates band compressions, allowing unambiguous sequence determination.

    Conclusions:

    • The modified T7 DNA polymerase is highly suitable for chain-termination DNA sequencing.
    • The enzyme's properties lead to increased accuracy, reduced background, and unambiguous sequence reads.
    • This modified polymerase represents a significant advancement for DNA sequencing technologies.