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Bio22 Test 3 Chap 9
Chapter9
| Question | Answer |
|---|---|
| Biotechnology | ♣ Biotechnology: The industrial application of microorganisms, cells, or cell components to make a useful product. |
| Explain why recombinant DNA practices are used for species that are unrelated. | ? |
| Define vector- two examples: | Two examples of a vector are plasmids and phages (viruses). |
| What is the role of a vector in providing a desired gene product? | A vector must be capable of replicating. Any DNA that is inserted in the vector will be replicated in the process. Thus, vectors serve as vehicles for the replication of desired DNA sequences. |
| Why is artificial selection important? (i.e adding ampicillin to media) | artificial selection is important to select desirable breeds of organisms with characteristics that enhance survival, and are more likely to survive and reproduce than variants that lack the desirable traits. |
| Examples of Artificial Selection | For example selecting an organism that is (ampR) Which is resistant to antibiotic ampicillin |
| What is site-directed mutagenesis? | Change a specific DNA code to change a protein |
| What is a restriction enzyme? | ♣ Restriction enzyme: An enzyme that cuts double-stranded DNA at specific sites between nucleotides. |
| What is the difference between sticky and blunt ends? | Blunt ends: are strands of DNA that are cut in the same place. |
| Sticky Ends? | ♣ Sticky ends: are most useful in rDNA because they can be used to join two different pieces of DNA that were cut by the same restriction enzyme |
| What is the restriction sequence of EcoR1 | the restriction sequence of EcoR1 is: AATTC and CTTAA |
| What is the rationale for using two different restriction enzymes when cutting a vector and desired gene? What happens when you cut both with the same enzyme? | ♣ when you cut foreign DNA and a vector with the same enzyme, the foreign DNA can then be inserted into the vector. |
| What is the polymerase chain reaction? | ♣ Polymerase chain reaction (PCR): A technique using DNA polymerase to make multiple copies of a DNA template in vitro |
| Describe the steps and temperatures of PCR.(Step 1-2) | 1.Incubate target DNA at 94°C for 1 minute to separate the strands. 2.Add primers, nucteotides (deoxynucteotides), and DNA potymerase |
| Describe the steps and temperatures of PCR.(Step 3-5) | 3.Primers attach to single-stranded DNA during incubation at 60°C for 1 minute. 4.Incubate at 72'C for 1 minute; during this time. two copies of target DNA are formed. 5.Repeat the cycle of heating and cooling to make two more copies of target DNA |
| What components go into a PCR mix? | •Primers (Short pieces of nucleic acid) •4 nucteotides (deoxynucteotides) (for assembly into new DNA) •DNA potymerase (enzyme |
| What is the relevance of Taq Polylmerase? | Thermlis aqllaticus (Taq Polylmerase): Is a thermophilic bacterium. The enzyme from such organisms can survive the heating phase without being destroyed. |
| How many new strands of DNA do you have after 5 PCR cycles? | 58 new strands of DNA can come from 1 strand of DNA after 5 PCR cycles. |
| What is transformation | Transformation: The process in which genes are transferred from one bacterium to another as “naked” DNA in solution |
| Why do you need competent cells for transformation? | Many cell types, including E. coli, yeast, and mammalian cells, do not naturally transform; however, simple chemical treatments can make all of these cell types competent, or able to take up external DNA. |
| How does heat and ice aid in transformation? | Competent cells are mixed with the cloned DNA and given a mild heat shock. Some of these cells will then take up the DNA. |
| What is electroporation? | Electroporation: A technique by which DNA is inserted into a cell using an electrical current. |
| Describe protoplast fusion | 1.Bacterial cell walls are enzymaticallydigested producing protoplasts. 2.In solution, protoplast are treated with polyethylene glycol. 3.Protoplast fuse. 4.Segments of the two chromosomes recombine. 5.Recombinant cell grows new cell walls. |
| How does a gene gun work | ♣ Microscopic particles of tungsten or gold are coated with DNA and propelled by a burst of helium through the plant cell walls. Some of the cells express the introduced DNA as though it were their own. |
| Which method is used to insert foreign DNA into animal cells? | Foreign DNA can be injected into animal cells by microinjection using a fine glass micropipette. |
| What is a genomic library? How is it built? Why is it important? | Genomic library: A collection of cloned DNA fragments created by inserting restriction enzyme fragments in a bacterium, yeast, or phage |
| What is cDNA? Why do we need it? How is it used in regards to genomic libraries (hint: eukaryotic cells)? | cDNA (complementary DNA): DNA made in vitro from an mRNA template by reverse transcriptase (enzyme). ♣ Molecules of cDNA produced from a mixture of all the mRNAs from a tissue or cell type can be cloned to form a cDNA library. ♣ |
| Part 2 | Complementary DNA (cDNA) made from mRNA by reverse transcription can be cloned in genomic libraries. |
| Part3 | In addition, if such a gene is put into a bacterial cell, the bacterium will not usually be able to remove the introns from the RNA transcript, |
| Part4 | and therefore it will not be able to make the correct protein product. However, an artificial gene that contains only exons can be produced by using an enzyme called reverse transcriptase to synthesize complementary DNA (cDNA) from an mRNA template |
| Part5 | The cDNA method is the most common method of obtaining eukaryotic genes. A difficulty with this method is that long molecules of mRNA may not be completely reverse-transcribed into DNA; the reverse transcription often aborts, forming only parts of the d |
| Part6 | A difficulty with this method is that long molecules of mRNA may not be completely reverse-transcribed into DNA; the reverse transcription often aborts, forming only parts of the desired gene |
| How is synthetic DNA used in PCR? | Some commercial products such as insulin, interferon, and somatostatin are produced from chemically synthesized genes |
| Explain how clones are selected using antibiotic resistance and blue-white screening. | ♣ The two genes, called marker genes, are used so that the insertion of plasmid DNA into the host bacterium can be determined. |
| Part1 | . In the blue-white screening procedure, a library of bacteria is cultured in a medium called X-gal. X-gal contains two essential components other than those necessary to support normal bacterial growth. |
| Part2 | One is the antibiotic ampicillin, which prevents the growth of any bacterium that has not successfully received the ampicillin-resistance gene from the plasmid |
| Part3 | The other, called X -gal, is a substrate for l3 -galactosidase. Only bacteria that picked up the plasmid will grow- because they are now ampicillin resistant. Bacteria that picked up the recombinant plasmid- |
| Part4 | in which the new gene was inserted into the IacZ gene- will not hydrolyze lactose and will produce white colonies. |
| Part6 | If a bacterium received the original plasmid containing the intact lacZ gene, the cells will hydrolyze X-gal to produce a blue-colored compound; the colony will be blue. |
| How are probes used to identify clones? | Single-stranded DNA that are complementary to the desired gene, are synthesized. If the DNA probe finds a match, it will adhere to the target gene. The DNA probe is labeled with a radioactive element or fluorescent dye so its presence can be determined. |
| What are the pros and cons of using E.coli versus yeast in producing gene products? | E. coli; is easily grown, and researchers are very familiar with this bacterium and its genetics |
| Part1-a | E. coli also has several disadvantages. Like othergram-negative bacteria, it produces endotoxins as part of the outer layer of its cell wall. |
| Part1-b | Because endotoxins cause fever and shock in animals, their accidental presence in products intended for human use would be a serious problem. |
| Part2 | It does not usually secrete protein products. To obtain a product, cells must usually be broken open (lysed) and the product purified from the resulting "soup" of cell components. Recovering the product is expensive when done on an industrial scale. |
| Part3-a | ♣ Yeast: (Saccharomyces cerevisiae) Its genome is only about four times larger than that of E. coli and is probably the best understood eukaryotic genome. |
| Part3-b | Yeasts may carry plasmids, and the plasmids are easily transferred into yeast cells after their cell walls have been removed. |
| Part3-c | As eukaryotic cells, yeasts may be more successful in expressing foreign eukaryotic genes than bacteria. Furthermore, yeasts are likely to continuously secrete the product. Because of all these factors, |
| Part3-d | yeasts have become the eukaryotic workhorse of biotechnology. |
| • Applications of rDNA How does rDNA aid in making vaccines? What is gene therapy? | Cells and viruses can be modified to produce a pathogen's surface protein, which can be used as a vaccine. |
| Part1 | DNA vaccines consist of rDNA cloned in bacteria. Gene therapy: 1.Treating a disease by replacing abnormal genes. 2.to replace Gene therapy can be used to cure genetic diseases by replacing the defective or missing gene. |
| What was the purpose of the Human Genome Project? | ♣ The goal of this project was to sequence (map) the entire human genome, approximately 3 billion nucleotide pairs, comprising 20,000 to 25,000 genes |
| What is DNA fingerprinting? What are some applications? | DNA fingerprinting: Analysis of DNA by electrophoresis of restriction enzyme fragments of the DNA. |
| What is bioinformatics? | ♣ Bioinformatics: The science of determining the function of genes through computer-assisted analysis. |
| Describe how a southern blot is formed | 1.DNA containing the gene of interest is extracted from human cells and cut into fragments by restriction enzymes. |
| Part2 | 2.The fragments are separated according to size by gel electrophoresis. |
| Part3 | 3.The fragments are put in a well at one end of a layer of agarose gel. Then an electrical current is passed through the gel. While the charge is applied, the different-sized pieces of DNA migrate through the gel at different rates |
| Part4 | 4.The separated fragments are transferred onto a nitrocellulose filter by blotting. |
| Part5 | 5. The filter is exposed to a radioactively labeled probe for a specific gene. The probe will base-pair (hybridize) with a short sequence present on the gene. |
| Part6 | 6. The filter is then exposed to X-ray film. The fragment containing the gene of interest is identified by a band on the developed film |
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