F08-BCH361-L16

F08-BCH361-L16 - Gene technology Dolly was the first cloned...

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Gene technology
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Dolly, was the first cloned vertebrate organism
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Fig B6 The cloning of a virus bacterial colonies are infected by a virus => the cells produce multiply copies of the virus which infect the neighboring cells => the bacterial cells are lysed and the cloned virus can be isolated from the plaques
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cloning of (human) DNA fragments into a bacterial or viral DNA by gene technology
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The cloning of the human DNA fragments with a viral vector Fig B8a
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Fig B8b The cloning of the human DNA fragments with a viral vector
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Fig B5a How to get the human DNA into the viral genome? use of restriction enodnucleases who cleave double-stranded DNA and produces “ sticky- ends”
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Different Restriction endonucleases have different cleaving sites
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An example: insertion of DNA by the use of restriction endonuclease EcoRI a) separation of the ends
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The foreign DNA (in green), which contains the same sticky ends is fused into the plasmid and the backbone is sealed by DNA Ligase
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How does bacteria protect their own DNA from degradation ? Methylation of endogenous DNA protects it from cleavage by its own restriction endonucleases.
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Photo of a DNA plasmid (small circular piece of DNA in bacteria) genetic engineering using plasmids
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How do you decide if the foreign DNA has been successfully inserted ? Plasmid contains selection markers (antibiotic resistance gene)
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Fig B10 The plasmid pBR322, one of the first widely used cloning vectors a cloning vector contains: -origin of replication - multiple cloning site - marker (for example antibiotics resistance gene)
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Genetic engineering: Expression of foreign proteins
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The aim of genetic engineering is - to produce a gene product in large quantities and/or - to create an organism with modified behavior A genetically engineered organism is intentionally changed at the molecular level so that it exhibits different traits
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Ethical concerns are common in the public and genetic engineering is subject to a controversial discussion. Genetic engineering on the level of the whole organism has been practiced by humans for thousands of years by selective breeding in agriculture. Mutants were induced by UV light, sun light (in earlier days) or chemicals. In contrast knowledge driven genetic engineering of individual genes has only been possible for the past 10 years. In contrast to conventional breeding, borderlines between species can be overcome by genetic engineering.
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There are two major fields of genetic engineering: A) the production of large amounts of proteins by bacteria B) the modification of plants in agriculture
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A) The expression of foreign proteins in bacteria Insulin was the first human protein which was produced by genetically modified bacteria
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There are problems which may arise and that have to be solved. For example, when eucaryotic proteins are expressed in E.coli : -introns have to removed -the Shine Dalgano Sequence has to be added to the mRNA - many eucaryotic genes undergo post-translational modifications, such as proteolytic cleavage,
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This note was uploaded on 09/30/2011 for the course BCH 361 taught by Professor Fromme during the Fall '08 term at ASU.

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F08-BCH361-L16 - Gene technology Dolly was the first cloned...

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