micro-chap26b - CHAPTER 26 BIOTECHNOLOGIES Biotechnology is...

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CHAPTER 26 BIOTECHNOLOGIES Biotechnology is the use of living organisms to carry out chemical processes for industrial or commercial application. Much of genetic engineering is based on molecular cloning , in which a double stranded DNA fragment from any source is recombined with a vector and introduced into a suitable host. Commonly employed cloning vectors include plasmids and bacteriophages. Specific biotechnological application depends not just on the ability to identify and clone a gene, but also on manipulating the expression of the gene to produce, identify and purify its protein product. Successful genetic engineering depends not only on being able to carry out molecular cloning but also on knowledge of replication, transcription, translation, and the regulatory aspects that control all of these processes. *Figure *Figure (cDNA synthesis for cloning) o Often, when one is preparing a gene for cloning and expression of the native protein, the mature mRNA has to be isolated, and then this needs to be converted into cNDA (complementary DNA) for cloning. o Reverse transcriptase forms a hairpin loop (folds back on itself) because it begins to copy a newly synthesized DNA copy. o Completion of cNDA cloning often yields very high levels of expression of mammalian genes in prokaryotes. The expressed gene is free of introns. Why is this important? Why do we clone cDNA? the introns are already spliced out so will give the correct protein after translation. *Figure o Finding a gene from its protein: reverse translation . To do this, one has to make a family of probes and perform nonstringent hybridization of restriction endonuclease cleaved fragments of host DNA to isolate the corresponding gene. Why? *Figure (Finding the right clone: transformant colonies growing on agar surface.
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o Methods to clone DNA include making gene libraries from total genomic DNA, or cloing a DNA fragment from the gene of interest. The fragment is often made by PCR using specific primers. If the gene is expressed, the presence of foreign protein, as detected by its activity or by reaction with specific antibodies, is evidence that the gene is present. However, if the gene is not expressed, its presence can be detected with a nucleic acid probe . For genes encoding small proteins, the entire gene could be synthesized for purposes of expression. Some proteins expressed in bacteria may not be expressed properly because they need to get post-translationally modified, and the enzymologies for some of these modifications are not present in bacteria. [Prokaryotes not capable of doing post- translation compared eukaryotes. The bacterial genomes do not encode the enzyme needed like it is on eukaryotes.] Some proteins may be insoluble in bacteria, and can be expressed but not isolated. These form inclusion bodies; from which the protein cannot often be recovered.
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This note was uploaded on 11/16/2010 for the course BIO 3317 taught by Professor Fentisel during the Spring '10 term at Temple.

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micro-chap26b - CHAPTER 26 BIOTECHNOLOGIES Biotechnology is...

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