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Chapter 3

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BIOL 342
Christine Dupont

Chapter 3- Recombinant DNA Technology Recombinant DNA Technology is also called “GENE CLONING”, this technology looks into the experimental protocols which lead to the transfer of DNA from one organism to another. Recombinant DNA Experiments - the DNA is extracted from a donor organism, is enzymatically cleaved and joined to another DNA (vector) in order to form a recombinant DNA molecule (aka DNA construct or Cloning vector) - the cloning vector is transferred into a HOST CELL, this process is called TRANSFORMATION - the transformed cells, which now have the DNA construct, can be identified and isolated - If needed, a DNA construct can be made so that the protein product encoded by the cloned DNA sequence is produced in the host cell Recombinant DNA Technology requires enzymes that recognize specific double stranded DNA sequences and cleave the DNA in both strands at these sequences.  Nucleases that cut internally = ENDONUCLEASES  Nucleases that degrade from the ends of nucleic acids = EXONUCLEASES RESTRICTION ENDONUCLEASES - For molecular cloning, BOTH the target DNA and the cloning vector need to be cut into discrete and reproducible fragments - The enzymes used are TYPE II RESTRICTION ENDONUCLEASES - One of the first type II RE’s was from E.coli, called EcoRI EcoRI will bind to the DNA region with a specific recognition site, and will cut to produce two single stranded complementary cut ends STICKY ENDS (Why sticky ends are good? any 2 complementary sticky ends, are capable of annealing) Restriction endonuclease cleavage is important in molecular cloning for inserting target DNA into a cloning vector. PLASMID CLONING VECTORS Plasmids are: - Self-replicating - Double stranded - Circular DNA - Maintained in bacteria as extra-chromosomal entities - Plasmids have the basics to make them vectors for carrying cloned DNA Plasmid Cloning Vector: pBR322 pBR322 is a general purpose plasmid cloning vector. It carries 2 antibiotic resistance genes. - one resistant to Amp Ampicillin - one resistant to Tet  Tetracycline It has unique BamHI, HindIII and SalII recognition sites all within the Tet gene Has unique PstI site  within the Amp gene Has an EcoRI site not within any coding DNA So how does the pBR322 Vector Work???? - The vectors are cut with a restriction enzyme that lies within either of the Amp or Tet genes and will cleave the plasmid DNA once to create a single, linear, sticky ended DNA - The linear molecules are combined with the target DNA of interest - This DNA has also been cut with the same restriction enzyme, creating the same sticky ends on the plasmid DNA - The DNA mix is then treated with ligase Transformation & Selection Once you introduce the target DNA into the vector, the next step is THE UPTAKE OF CLONED PLASMID DNA by a bacterial cell (usually E.coli)  this process is called TRANSFORMATION 2 Parameters to measure the success of transformed cells: TRANSFORMATION FREQUENCY: is the ratio of transformed cells to the total number of treated cells TRANSFORMATION EFFICIENCY: is the number of transformed cells in comparison to the DNA that was originally added to the cells Following the transformation step, we want to identify the cells that contain plasmids with cloned DNA (constructs) from those that weren’t able to be transformed and uptake the DNA inserts. Vector = alone….. Vector + Insert= Construct So with pBR322… if the Target DNA was inserted into the BamHI site, the 2 antibiotic resistance markers are used to identify this. - Then the mixture is plated onto medium containing antibiotic, ampicillin  mixture contains 1) non-transformed cells 2) cells with plasmid 3)cells with DNA cloned into BamHI site of pBR322 - Cells which CARRY THE VECTOR with or without the insert can grow in the medium; the NONTRANSFORMED CELLS are sensitive to ampicillin  will not grow because they do not have ampicillin resistance  We know the BamHI site of pBR322 is within the Tet gene, so inserting DNA into this gene will disrupt the coding sequence and Tet resistance is lost (gene is not expressed).  At this point, cells with the construct are RESISTANT to amp and sensitive to Tet  Cells with plain vector (no insert) pBR322 DNA will have an intact Tet gene and are resistant to both amp and Tet  Second step: to distinguish between these 2 cells which only carry the vector, and cells which carry the insert (construct containing the target DNA) Cells that grow on amp-containing medium are then plated to a Tet-containing medium Cells that form colonies on the Tet-plates carry pBR322 w
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