Chapter_20_Outline - Chapter 20: DNA Technology and...

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Chapter 20: DNA Technology and Genomics I. DNA Cloning 1) Advances in recombinant DNA (DNA in which genes from two different sources are combined in vitro into the same molecule) technology have helped scientists study the eukaryotic genome by allowing genetic engineering (direct manipulation of genes for practical purposes) to take place. Scientists can make recombinant DNA and introduce it to cultured cells that replicate the DNA to yield a desired protein. DNA technology is applied in areas ranging from agriculture to criminal law. 2) Restriction enzymes (enzymes that cut DNA molecules at a limited number of specific locations) protect the bacteria against intruding DNA from other organisms, such as phages or other bacterial cells. 3) The creation of sticky ends (restriction fragments are double-stranded DNA fragments with at least one single-stranded end, the sticky end) by restriction enzymes is useful in producing a recombinant DNA molecule because these short extensions will form hydrogen-bonded base pairs with complementary single-stranded stretches on other DNA molecules cut with the same enzyme. 4) Procedures for cloning a eukaryotic gene in a bacterial plasmid: 1. isolate plasmid (vector) DNA and human DNA 2. insert human DNA into plasmids a. cut both DNAs with same restriction enzyme b. mix the DNAs; they join by base pairing c. add DNA ligase to bond covalently 3. put plasmids into lacZ - bacteria by transformation 4. clone cells a. plate cells onto medium with ampicillin and X-gal b. identify clones of cells containing recombinant plasmids by their ability to grow in presence of ampicillin and their white color 5. identify clone carrying gene of interest 5) Expression vectors allow the synthesis of many eukaryotic proteins in bacterial cells. It contains the requisite prokaryotic promoter just upstream of a restriction site where the eukaryotic gene can be inserted. 6) Fully processed mRNA, which has been stripped of introns in the eukaryotic cell nucleus can be extracted from cells and then use the enzyme reverse transcriptase (obtained from retroviruses) to make DNA transcripts of this RNA. Each DNA molecule produced carries the complete coding sequence for a gene but no introns. 7) They are as easy to grow as bacteria, and they have plasmids, a rarity among eukaryotes. 8) Three ways to aggressively introduce recombinant DNA into eukaryotic cells are electroporation (applying a brief electrical pulse to a solution containing cells to create a temporary hole in the cell’s plasma membrane, through which DNA can enter), injecting DNA directly into single eukaryotic cells using microscopically thin needles, and attaching DNA to microscopic particles of metal and firing the particles into cells with a gun. 9)
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Chapter_20_Outline - Chapter 20: DNA Technology and...

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