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Spontaneous insertions into cosmid vector: a warning.

C Fernandez, D Larhammar, B Servenius

    Gene
    |January 1, 1986
    PubMed
    Summary
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    Instability in pNNL cosmid clones, including deletions and rearrangements, was linked to the presence of Escherichia coli insertion elements IS1 within the vector's cos region, impacting human genomic library characterization.

    Area of Science:

    • Molecular Biology
    • Genomics
    • Bacteriology

    Background:

    • Characterization of human genomic libraries is crucial for genetic research.
    • Cosmid vectors like pNNL are commonly used for constructing genomic libraries.
    • Vector instability can hinder the accurate representation and analysis of genomic inserts.

    Purpose of the Study:

    • To investigate the causes of instability observed in pNNL cosmid clones from a human genomic library.
    • To identify the specific DNA sequences responsible for deletions and rearrangements in the vector and inserts.
    • To understand the implications of these findings for genomic library construction and maintenance.

    Main Methods:

    • Restriction mapping of rearranged pNNL cosmid clones.
    • Nucleotide sequencing of the affected vector region.

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  • Bioinformatic analysis to identify known DNA elements within the sequenced region.

    • Main Results:

    • Observed instability in pNNL cosmid clones, including short inserts, deletions, and vector rearrangements.
    • Identified deletions in the cos region of the pNNL vector, specifically between ClaI and HindIII sites.
    • Sequencing revealed the presence of Escherichia coli insertion elements IS1 in the unstable vector segments.

    Conclusions:

    • The instability of pNNL cosmid clones is attributed to the presence of Escherichia coli IS1 insertion elements.
    • IS1 elements in the cos region of the pNNL vector lead to deletions and rearrangements, compromising library integrity.
    • These findings highlight the importance of considering insertional elements in vector design for stable genomic library construction.