-Is the process of manipulating genes and genomes

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1 Genetic Engineering -Is the process of manipulating genes and genomes Biotechnology -Is the process of manipulating organisms or their components for the purpose of making useful products Restriction Enzymes Recombinant DNA -Are used to cut strands of DNA at specific locations (called restriction sites). They are derived from bacteria -When a DNA molecule is cut by restriction enzymes, the result will always be a set of restriction fragments which will have at least one single-stranded end, called a sticky-end -Sticky ends can form hydrogen bonds with complementary single-stranded-pieces of DNA these unions can be sealed with the enzyme DNA ligase -Is DNA that has been artificially made, using DNA from different sources and often different species. An example is the introduction of a human gene into an E coli bacterium

2 Gene Cloning -Is the process by which scientists can produce multiple copies of specific segments of DNA they can then work with in the lab Plasmid Step One of Gene Cloning -Extra small circular double stranded pieces of FNA found in viral, yeast, or bacterial cells used extensively in Biotechnology and Recombinant DNA -Serve as a vehicle for transporting genes from one organism into another (transformation) so multiple copies can be made (gene cloning) -Gene cloning yields multiple copies of a gene or other DNA segment -The cloning of genes generally occurs in five steps -Identify and isolate the gene of interest and a cloning vector. The vector is the plasmid (usually bacterial) that will carry the DNA sequence to be cloned Step Two of Gene Cloning -Cut both the gene of interest and the vector with the same restriction enzyme. This gives the plasmid and the human gene matching sticky ends

3 Step Three of Gene Cloning -Join the two pieces of DNA from recombinant plasmids by mixing the plasmids with the DNA fragments. The human DNA fragments can be sealed into the plasmid using DNA ligase Step Four of Gene Cloning -Get the plasmid vector carrying the gene of interest into a host cell. The plasmids are taken up by bacterium by transformation Step Five of Gene Cloning Application of Gene Cloning -Select for cells that have been transformed. The bacterial cells carrying the clones must be identified or selected -This can be done by linking the gene to an antibiotic resistance gene or a reporter gene such as green fluorescent protein. -In the transformation lab, we will use an ampicillin-resistant plasmid. Any bacterial cells that did not pick up the plasmid by transformation will be killed when grown on agar with the antibiotic ampicillin -Insulin Production -Human Growth Hormone Production -Production of other proteins that make crops resistant to pests

4 Comments about Recombinant Plasmid Another Biotechnology Tool We Use is PCR -Gene can t be above certain size or a plasmid won t work -mrna must not need splicing to remove introns because we are placing it directly into a cell to be expressed (bacteria can t remove introns) -But if we still want a bacterial cell to make a human protein that does need mrna processing, we can make cdna copy of the gene and insert that instead -cdna is created using reverse transcriptase to turn a processed mrna coding for a certain protein back into a DNA to insert into the bacterial plasmid -PCR (polymerase chain rxn) is a method used to greatly amplify a particular piece of DNA without the use of cell -PCR is used to amplify DNA when the source is impure or scanty (as it would be at a crime scene) -DNA technology allows us to study the sequence, expression, and function of a gene Gel Electrophoresis Forensic Applications -Is a lab technique that is used to separate macromolecules, primarily DNA and proteins, on the basis of their size and charge with the use of an electrical current -In separating DNA, the negative charges on phosphates in the molecule cause DNA to move more toward the positive pole -The gel allows smaller molecules to move more easily than larger fragments of DNA. The DNA fragments are separated by size -Links suspect bodily to the crime scene, but doesn t prove they committed the crime -Results take much longer then on TV shows -Analysis of old evidence is reversing some sentences

5 Cloning organisms may lead to production of stem cells for research and other applications Stem Cells -In animal cloning the nucleus of an egg is removed and replaced with the diploid nucleus of a body cell, a process termed nuclear transplantation -The ability of a body cell to successfully form a clone decreases with embryonic development and cell differentiation -The major goal of most animal cloning is reproduction, but not for humans -In humans, the major goal is the production of stem cells -Can both reproduce itself indefinitely and under the proper conditions, produce other specialized cells. Stem cells have enormous potential for medical applications Diagnosis of Disease Gene Therapy -If the sequence of a particular virus s DNA or RNA is known, PCR can be used to amplify patient s blood samples to detect even small traces of the virus -Different alleles have different DNA sequences --These different sequences can be found using restriction enzymes that yield different lengths of DNA fragments, or restriction fragment length polymorphisms (RFLP) -The difference in banding patterns after electrophoresis allows for diagnosis of the disease, or even a carrier of the disease -The alteration of an afflicted individual s genes. Gene therapy holds a great potential for treating disorders traceable to a single defective gene, such a cystic fibrosis

6 The Production of Pharmaceuticals -Gene splicing and cloning can be used to produce large amounts of particular proteins in the lab Forensic Applications -DNA samples taken from the blood, skin cells, or hair of alleged criminal suspects cen be amplified with PCR and compared to DNA collected from the crime scene DNA fingerprints (electrophoretic bands that are unique to each individual) can be compared and used to identify persons at that crime scene Environmental Cleanup -Scientists engineer metabolic capabilities into microorganisms, which are then used to treat environmental problems, such as removing heavy metals from toxic mining sites or engineering bacteria to digest oil Agricultural Applications -Certain genes that produce desirable traits have been inserted into crop plants to increase their productivity and efficiency. We have genetically engineered rice to contain vitamin A, saving about 60,000 children in Asia per year from death due to this deficiency -An organism that has acquired by artificial means one or more genes from another species or variety is termed a genetically modified organism (GM organism)