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Cloning - Cloning Genes

dna human insulin figure

Molecular cloning is performed to enable researchers to have many copies of genetic material available in the laboratory for the purpose of experimentation. Cloned genes allow researchers to examine encoded proteins and are used to sequence DNA. Gene cloning also allows researchers to isolate and experiment on the genes of an organism. This is particularly important in terms of human research; in instances where direct experimentation on humans might be dangerous or unethical, experimentation on cloned genes is often practical and feasible.

Cloned genes are also used to produce pharmaceutical drugs, insulin, clotting factors, human growth hormone, and industrial enzymes. Prior to the widespread use of molecular cloning, these proteins were difficult and expensive to manufacture. For example, before recombinant DNA technology, insulin (a pancreatic hormone that regulates blood glucose levels) used by people with diabetes was extracted and purified from cow and pig pancreases. Since the amino acid sequences of insulin from cows and pigs are slightly different than those in human insulin, some patients experienced adverse immune reactions to the nonhuman "foreign insulin." FIGURE 8.1
Bacterial plasmid
SOURCE: "Bacterial Plasmid," in Cloning Fact Sheet, U.S. Department of Energy, Office of Biological and Environmental Research, Human Genome Project, 2004, http://www.ornl.gov/sci/techresources/Human_Genome/elsi/cloning.shtml (accessed February 24, 2005)
FIGURE 8.2
Standardized clone names
SOURCE: "Standardized Clone Names," in "Clone Nomenclature," NCBI Clone Registry U.S. Department of Health and Human Services, National Institutes of Health, National Center for Biotechnology Information, National Library of Medicine, http://www.ncbi.nlm.nih.gov/genome/clone/nomen.html (accessed February 24, 2005)
The recombinant human version of insulin is identical to human insulin so it does not produce an immune reaction.

Figure 8.3 shows how a gene is cloned. First, a DNA fragment containing the gene being studied is isolated from chromosomal DNA using restriction enzymes. It is joined with a plasmid (a small ring of DNA found in many bacteria that can carry foreign DNA) that has been cut with the same restriction enzymes. When the fragment of chromosomal DNA is joined with its cloning vector (cloning vectors, such as plasmids and yeast artificial chromosomes, introduce foreign DNA into host FIGURE 8.3
Cloning DNA in plasmids
SOURCE: "Cloning DNA in Plasmids," U.S. Department of Energy, Office of Biological and Environmental Research, Human Genome Project, http://www.ornl.gov/…/graphics/slides/images2.shtml (accessed February 25, 2005)
cells), it is called a recombinant DNA molecule. Once it has entered into the host cells, the recombinant DNA can be reproduced along with the host cell DNA.

Another molecular cloning technique that is simpler and less expensive than the recombinant cloning method is the polymerase chain reaction (PCR). PCR has also been dubbed "molecular photocopying" because it amplifies DNA without the use of a plasmid. Figure 8.4 FIGURE 8.4
Polymerase chain reaction
SOURCE: "Polymerase Chain Reaction," in Talking Glossary of Genetic Terms, U.S. Department of Health and Human Services, National Institutes of Health, National Human Genome Research Institute, http://www.genome.gov/Pages/Hyperion//DIR/VIP/Glossary/Illustration/PCR.shtml (accessed February 25, 2005)
shows how PCR is used to generate a virtually unlimited number of copies of a piece of DNA.

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