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• Outline the steps in gene cloning, including predicted outcomes from successful versus unsuccessful steps. • Explain the role of a vector in molecular cloning. • Relate endogenous roles of enzymes to their recombinant DNA applications. • Compare and contrast the construction of genomic and cDNA libraries. • Explain why DNA fragments can be separated with gel electrophoresis. • Compare endogenous DNA replication with sequencing and with the polymerase chain reaction.
20.2 Genomics
• Explain the difference between ‘genomics’ and ‘functional genomics’. • Describe the steps involved in in vitro DNA synthesis techniques illustrated in the chapter (PCR, dideoxy sequencing). • Explain how DNA microarrays can be used to identify the genes expressed by an organism. • Explain the value of DNA microarrays to biological and biomedical research.
20.3 Biotechnology
• Provide examples of medically important eukaryote proteins that are produced in bacterial cells using biotechnology. • Explain how a human protein can be produced by the cells of a sheep. • Describe three applications of cloning technology. • Describe a genetically modified organism (GMO) organism. • Compare and contrast a transgenic organism with an organism that is the result of gene knock-out manipulation. • Compare recombinant technology techniques in plants with those in bacteria. • Differentiate between gene cloning and reproductive cloning.
Chapter Summary
Science has reached the exciting, but potentially dangerous stage, at which we are learning to manipulate the materials of heredity. The first human genes isolated and inserted into bacteria turned these cells into miniature factories producing interferon. Many bacteria possess restriction endonucleases to protect themselves from invading viruses. Scientists use these enzymes to chop up strands of DNA at specific locations. Such specificity assures that a given enzyme will always break up a specific kind of DNA into exactly the same size and number of fragments. These fragments constitute a library of DNA sequence information. Restriction enzyme specificity also assures that all of the fragments possess identical, short sequences called “sticky ends.” Each strand of a sticky end is complementary to the other strand and can be joined to the other ends when treated with a DNA ligase. DNA Fragments, even those from different organisms, that have been cut with the same restriction enzyme can be joined enabling the insertion of foreign genes into a plant, animal, or bacterial genome.
Bacterial plasmids and viruses are the vehicles by which such genes are inserted into the host DNA, the crux of genetic engineering. There are four steps in this process: cleavage, producing recombinant DNA, cloning, and screening. Cleavage is accomplished using the restriction endonuclease that will produce the desired sticky ends. The fragments are then inserted into the desired vehicle. Unfortunately, very few vehicles actually receive DNA fragments and even fewer get the desired piece. At this point, vehicles not carrying fragments are eliminated, generally by prior association with an antibiotic resistance gene. Each colony of cells is cloned and allowed to multiply, thus replicating not only its own genome but the added fragment as well. The clones are then screened to determine which clonal line contains the desired fragment.
Polymerase chain reaction is another new molecular technique that amplifies DNA in an in vitro sample. Frequently the DNA in a sample (of blood for example) is so small that it cannot be analyzed directly. With PCR, the DNA is copied using a microprocessor-controlled thermoregulator. The DNA unzips as the temperature is increased. When it is lowered, polymerase enzymes catalyze the replication of DNA from special primers, making a new strand from each original strand. Thus the amount of DNA is doubled at each cycle – 2 strands to 4 strands to 8 strands to 16 strands and so forth. This method is substantially quicker than cloning the DNA strand via plasmids or viruses. DNA is readily identified using a technique called Southern blotting. Differences in DNA sequences are identified by RFLP analysis. Each individual can be identified by the RFLP patterns possessed, what is referred to as a DNA fingerprint.
Biotechnology uses genetic engineering techniques to solve practical problems. The biological community is busy sequencing the entire human genome, certainly an enormous task. DNA fingerprinting has been used to identify and convict numerous criminals. Dozens of commercial applications exist to utilize this revolutionary technology. The most obvious application, pharmaceuticals, however, encounters additional problems of separating the desired product from the rest of the cellular material. Attempts are being made to construct piggyback vaccines, placing genes coding for the exterior of a virulent virus within the harmless vaccinia virus. Agricultural uses range from developing resistance to herbicides, viruses, and insects; to inserting genes for nitrogen fixation and improving growth and plant nutritional value.
Society must be informed about these biological processes to ensure our safety and economic wellbeing, as well as that of future generations. Lack of sufficient biological knowledge is the source of most of the public’s concern about genetically engineered products. Many assume that BST in milk products may cause human growth problems; they lack the physiological knowledge that this protein is degraded in the stomach like all other proteins. A great many people do not trust governmental safeguards and fear the inadvertent or intentional development of lethal viruses and bacteria. Although there is little scientific need for labeling genetically modified food products, the public has the right to insist upon it. If properly done, labeling should serve to educate consumers as well as inform them.
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