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Eukaryotic gene cloning/expression, PCR, gel electrophoresis, Southern blotting, DNA sequencing, gene expression/function, cloning

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Colorado State University
Life Science
LIFE 102
Louis Bjostad

27 AprilExpressing Cloned Eukaryotic GenesAfter a gene has been cloned its protein product can be produced in larger amounts for researchCloned genes can be expressed as protein in either bacterial or eukaryotic cellsBacterial Expression SystemsSeveral technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cellsTo overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active prokaryotic promoterEukaryotic Cloning and Expression SystemsThe use of cultured eukaryotic cells as host cells and yeast artificial chromosomes YACs as vectors helps avoid gene expression problemsYACs behave normally in mitosis and can carry more DNA than a plasmidEukaryotic hosts can provide the posttranslational modifications that many proteins requireOne method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranesAlternatively scientists can inject DNA into cells using microscopically thin needlesOnce inside the cell the DNA is incorporated into the cells DNA by natural genetic recombinationAmplifying DNA in Vitro The Polymerase Chain Reaction PCRThe polymerase chain reaction PCR can produce many copies of a specific target segment of DNAA threestep cycleheating cooling and replicationbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
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