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Chapter 18 Textbook Notes

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BIOL 102
Daniel Lefebvre

Genetic Technology Gene Cloning: • Purpose: o Produce large amounts of DNA of a specific gene o Express the cloned gene to produce the encoded protein • Reproductive cloning – cloning of multicellular organisms 1. Vector DNA and Chromosomal DNAAre the Starting Materials to Clone a Gene • Vector – type of DNA molecule which can carry a small segment of chromosomal DNA o Plasmids – small, circular pieces of DNA o viral vector – infect living cells and self-reproduce by taking over the host cell’s metabolic machinery 2. Cutting Chromosomal and Vector DNA into Pieces and Linking Them Together Produces Recombinant DNA molecules • Restriction enzyme – cuts enzyme; bind to a specific base sequence and then cleaves DNA backbone in two defined locations, one in each strand • Restriction sites – the sequences recognized by restriction enzyme • Sticky ends – used by restriction enzymes to digest DNA into fragments • Recombinant vector/hybrid vector –results when a fragment of chromosomal DNA can become ligated to both ends of the vector 3. Putting Recombinant Vectors into Host Cells and Allowing Those Cells to Propagate Achieves Genetic Cloning • Selectable marker – a gene inserted into a plasmid vector, where the vector already carries an antibiotic resistance gene (amp gene) • Transformation – when a plasmid vector is used in the cloning of the actual gene of interest o Single bacterial cell usually takes up a single recombinant vector • Transfection – when a viral vector is introduced into a cell in the cloning of the actual gene of interest • DNA library – collection of recombinant vectors, with each vector containing a particular fragment of chromosomal DNA (chromosomal DNA +restriction enzyme = tons of different DNA fragments, which are then ligated to form the recombinant vectors) o Genomic library – when inserts are derived from chromosomal DNA and represent the entire genome of the organism. (ex. Genomic libraries prepared from mouse liver or brain tissues will be essential identical) o Complementary DNA (cDNA) – recombinant vectors have cDNA inserts, which represent all the genes expressed in the original sample. (cDNA cloned from liver and brain tissues are composed of clones that are similar) o Colony hybridization – a method of identifying which colony contains the gene of interest, where a researcher uses a probe to identify which colony has it  Filter laid onto master plate and the lifted, yielding a replica of master plate  Lifted filter is treated with detergent to make bacterial permeable and the DNA is fixed to the filter  DNA is then denatured into single strands and radioactively labeled probe is added that is complementary to the gene of interest  Filter is placed in X-ray film and is then matched up with the master plate Southern Blotting – can detect presence of a particular gene within a mixture of many chromosomal fragments separated on a gel • Basis is that two DNA fragments will bind to each other only if they have complementary sequences • DNA is isolated and digesting using a restriction enzyme • Resulting chromosomal pieces are loaded onto gel which separates them according to mass Gel electrophoresis – technique that is used to separate macromolecules on a gel • Electric field used, causing charged molecules to migrate from top to bottom of gel o Sorts by charge (DNA is negatively charged and will move toward positive end/bottom) o Size/length/mass (smaller, lighter fragments also move mo
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