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A system for shotgun DNA sequencing.

J Messing, R Crea, P H Seeburg

    Nucleic Acids Research
    |January 24, 1981
    PubMed
    Summary
    This summary is machine-generated.

    Researchers created M13mp7, a modified phage for cloning DNA fragments. This new tool simplifies DNA sequencing and genetic manipulation, enhancing molecular biology research.

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    Area of Science:

    • Molecular Biology
    • Genetics
    • Biotechnology

    Background:

    • The M13mp2 phage contains the beta-galactosidase gene, crucial for genetic studies.
    • Existing methods for DNA cloning and sequencing had limitations in efficiency and scope.

    Purpose of the Study:

    • To engineer a novel M13 phage vector (M13mp7) with enhanced cloning capabilities.
    • To facilitate direct cloning of DNA fragments generated by various restriction endonucleases.
    • To streamline the process of nucleotide sequence determination for cloned DNA.

    Main Methods:

    • Introduction of a multipurpose cloning site into the beta-galactosidase gene of M13mp2 using synthetic DNA.
    • Modification of viral gene II by single base-pair mutations to remove restriction endonuclease cleavage sites.

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  • Utilizing chain terminators and a synthetic oligonucleotide primer for rapid DNA sequencing.

    • Main Results:

    • The new M13mp7 phage allows direct cloning of DNA fragments from diverse restriction digests.
    • The introduced cloning site adds 14 codons without impairing lac gene complementation.
    • Nucleotide sequences of cloned DNA are rapidly determined using the developed sequencing method.

    Conclusions:

    • M13mp7 represents a significant advancement in molecular cloning vectors.
    • The engineered phage simplifies DNA fragment cloning and sequencing, accelerating genetic research.
    • This tool enhances the ability to analyze and manipulate DNA sequences efficiently.