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A cosmid vector that facilitates restriction enzyme mapping.

P F Little, S H Cross

    Proceedings of the National Academy of Sciences of the United States of America
    |May 1, 1985
    PubMed
    Summary
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    Researchers developed a stable cosmid vector, loric, for efficient DNA packaging. This new "phosmid" system enables rapid restriction mapping of large DNA fragments, demonstrated on mouse chromosome 17.

    Area of Science:

    • Molecular Biology
    • Genomics
    • Recombinant DNA Technology

    Background:

    • Cosmid vectors are essential tools for cloning large DNA fragments.
    • Existing ColE1-derived cosmids exhibit instability issues.
    • Phage lambda origin of replication offers potential for improved vector stability.

    Purpose of the Study:

    • To construct and characterize a novel cosmid vector, loric, with enhanced stability.
    • To develop an efficient method for packaging loric recombinants into phosmid molecules.
    • To establish a rapid restriction mapping technique for phosmids.

    Main Methods:

    • Construction of the loric cosmid vector derived from the phage lambda origin of replication.
    • In vivo packaging of loric recombinants to produce phosmids.

    Related Experiment Videos

  • Labeling of cos sites (cos-L or cos-R) using 32P-labeled oligonucleotides.
  • Partial restriction enzyme digestion, agarose gel electrophoresis, and autoradiography for mapping.

    • Main Results:

    • Loric demonstrates greater stability compared to ColE1-derived cosmids.
    • Efficient in vivo packaging yields 100-300 micrograms of linear phosmid DNA per liter.
    • Rapid restriction mapping of the H-2 locus on mouse chromosome 17 was successfully achieved.

    Conclusions:

    • The loric vector provides a stable and efficient system for cloning and manipulating large DNA fragments.
    • Phosmid technology facilitates rapid and accurate restriction mapping of complex genomes.
    • This approach is valuable for genomic research, particularly in mapping specific loci like the H-2 locus.