Recombineering linear BACs

Qingwen Chen, Kumaran Narayanan

    Research output: Chapter in Book/Report/Conference proceedingChapter (Book)Researchpeer-review

    Abstract

    Recombineering is a powerful genetic engineering technique based on homologous recombination that can be used to accurately modify DNA independent of its sequence or size. One novel application of recombineering is the assembly of linear BACs in E. coli that can replicate autonomously as linear plasmids. A circular BAC is inserted with a short telomeric sequence from phage N15, which is subsequently cut and rejoined by the phage protelomerase enzyme to generate a linear BAC with terminal hairpin telomeres. Telomere-capped linear BACs are protected against exonuclease attack both in vitro and in vivo in E. coli cells and can replicate stably. Here we describe step-by-step protocols to linearize any BAC clone by recombineering, including inserting and screening for presence of the N15 telomeric sequence, linearizing BACs in vivo in E. coli, extracting linear BACs, and verifying the presence of hairpin telomere structures. Linear BACs may be useful for functional expression of genomic loci in cells, maintenance of linear viral genomes in their natural conformation, and for constructing innovative artificial chromosome structures for applications in mammalian and plant cells.
    Original languageEnglish
    Title of host publicationBacterial Artificial Chromosomes
    EditorsKumaran Narayanan
    Place of PublicationNew York NY USA
    PublisherHumana Press
    Pages27 - 54
    Number of pages28
    Edition2nd
    ISBN (Print)9781493916511
    DOIs
    Publication statusPublished - 2015

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