Scribd Logo
Upload

Welcome to Scribd!

  • 2 views

    Red-Gam TAR

    Uploaded by

    dasaniket830

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd

    Red-Gam TAR

    Uploaded by

    dasaniket830
    2 views6 pages

    Copyright

    © © All Rights Reserved

    Available Formats

    PDF, TXT or read online from Scribd

    Did you find this document useful?

    Is this content inappropriate?

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    2 views6 pages

    Red-Gam TAR

    Uploaded by

    dasaniket830

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 6

    The lambda red system is derived from the lambda red

    bacteriophage and its use as a genetic engineering tool is


    frequently called recombineering - short for homologous
    recombination-mediated genetic engineering.

    It allows for direct modification of DNA within E. coli and is


    independent of restriction sites.
    The lambda red recombineering system has three components.
    Gam: Gam prevents both the endogenous RecBCD and SbcCD nucleases from digesting
    linear DNA introduced into the E. coli.
    Exo: Exo is a 5’→3’ dsDNA-dependent exonuclease. Exo will degrade linear dsDNA
    starting from the 5’ end and generate 2 possible products: 1) a partially dsDNA duplex
    with single-stranded 3’ overhangs or 2) if the dsDNA was short enough, a ssDNA
    whose entire complementary strand was degraded.
    Beta: Beta protects the ssDNA created by Exo and promotes its annealing to a
    complementary ssDNA target in the cell.
    1. Substrate DNA design and
    generation
    2. Expression of lambda red
    recombination genes.
    3. Electroporation of substrate DNA
    and outgrowth of bacteria.
    4. Selection and confirmation of
    recombinant clones.
    TAR

    Transformation-associated recombination (TAR) cloning


    represents a unique tool for isolation and manipulation of large
    DNA molecules. The technique exploits a high level of
    homologous recombination in the yeast Sacharomyces
    cerevisiae. So far, TAR cloning is the only method available to
    selectively recover chromosomal segments up to 300 kb in
    length from complex and simple genomes. In addition, TAR
    cloning allows the assembly and cloning of entire microbe
    genomes up to several Mb as well as engineering of large
    metabolic pathways.
    The consecutive experimental steps of selective gene isolation. Step 1: The diagram
    shows TAR cloning of a gene of interest from total genomic DNA with a TAR vector
    containing YAC and BAC cassettes and two unique targeting sequences (hook1 and
    hook2) (in green) homologous to the 5′ and 3′ end of a gene of interest. The size of
    hooks may be as small as 60 bp (Noskov et al. 2001). The hook sequences may either
    be both unique, or one of the hook sequences may be a common repeat (for
    example, an Alu repeat for cloning from human genomic DNA). The TAR vector DNA
    is linearized by a unique endonuclease located between the hooks to expose
    targeting sequences. If necessary, genomic DNA may be treated by CRISPR/Cas9
    endonuclease before yeast transformation, which will greatly increase the yield of
    gene/region-positive TAR clones. Step 2: Genomic DNA and a linearized TAR vector
    are cotransformed into yeast Saccharomyces cerevisiae cells. Steps 3 and 4:
    Recombination between targeting sequences in the vector and the targeted
    sequences in the genomic DNA fragment leads to rescue of the fragment (or gene) as
    a circular TAR/YAC/BAC molecule. Step 5: Transferring TAR-isolated molecules
    containing a region of interest from yeast cells to bacterial cells by electroporation.
    Step 6: BAC DNA is isolated by a standard procedure for further sequencing or
    functional analysis

    You might also like