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Coliphage M13 cloning system and ddXTP chain-termination method for DNA sequencing

Y P Qi

    Scientia Sinica. Series B, Chemical, Biological, Agricultural, Medical & Earth Sciences
    |June 1, 1986
    PubMed

    Insights

    Researchers sequenced yeast mitochondrial DNA fragments using M13 cloning and Sanger sequencing. Optimizing the insert/vector ratio enhanced recombination frequencies for efficient DNA sequencing.

    Area of Science:

    • Molecular Biology
    • Genetics
    • Biochemistry

    Background:

    • Mitochondrial DNA sequencing is crucial for understanding cellular respiration and genetic diversity.
    • Efficient methods for DNA fragment isolation and sequencing are essential in molecular biology research.

    Purpose of the Study:

    • To sequence two specific DNA fragments of the cytochrome b gene from yeast mitochondrial DNA.
    • To optimize the M13 cloning and Sanger sequencing methodology for improved efficiency and accuracy.

    Main Methods:

    • Utilized Messing's M13 cloning system with M13mp8 and M13mp9 vectors.
    • Employed Sanger's dideoxynucleotide triphosphate (ddXTP) chain-termination method for sequencing.
    • Transfected competent Escherichia coli JM103 cells with recombinant DNA constructs.

    Main Results:

    • Successfully sequenced two yeast mitochondrial DNA fragments (575 bp and 709 bp).
    • Achieved high frequencies of recombination and positive recombination by maintaining a 3:1 insert/vector ratio.
    • Determined that reducing the ddXTP/dXTP ratio is necessary for reading longer DNA sequences, with 394 bp read on a single X-ray film.

    Conclusions:

    • The M13 cloning system and Sanger sequencing are effective for analyzing yeast mitochondrial DNA.
    • Optimizing the insert/vector ratio is critical for maximizing recombination efficiency.
    • Adjusting nucleotide ratios in Sanger sequencing can enhance the read length of DNA fragments.

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