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What are restriction enzymes and how do they work?
Restriction enzymes are bacterial enzymes that recognize a specific sequence (restriction site) and cut both strands of DNA at that sequence. Most recognition sites are a palindrome (RCCR).
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What is the difference between staggered end cut and blunt end cuts made by restriction enzymes?
- Staggered-end cuts: have single-stranded overhangs. These can base pair with complementary staggered-end cuts cut using the same restriction enzyme and anneal.
- Blunt-end cuts: have double stranded ends. These do not have an area for hybridization.
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How is DNA fragmented and separated on gel following restriction enzyme treatment?
- Restriction mapping: Establishes the number of, order of, and distances between restriction-enzyme cleavage sites along a cloned segment of DNA.
- Created by cutting DNA with different restriction enzymes and separating by gel electrophoresis (smallest fragments moves farthest through gel) creating a unique series of bands.
- Southern blotting: 1) separation of DNA fragments by gel electrophoresis 2) hybridization using labeled probes.
- Northern blotting: mRNA blotting (actively expressed genes)
- Western blotting: analyzing proteins
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What are RFLPs?
restriction fragment length polymorphisms: variations in the short base sequences where restriction enzymes can cut. Comparisons can be made between two samples cut with the same restriction enzyme
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What are the differences between these vectors, and what hosts would they be introduces to? plasmid, YAC, BAC, HAC, Ti Plasmid, Cosmid
- Plasmid: introduced to bacteria via transformation. Plamsid and DNA are cut with same restriction enzyme, then allowed to anneal. Many copies per bacteria. Blue-white selection is used to determine which bacteria have the desired product. Too small for some larger genes.
- YAC: yeast artificial chromsomes. Can clone large-fragments of DNA. Linear, has telomers, origins or rep, centromere.
- BAC: bacterial articificial chromsomes. Large, but low copy (1-2 per cell) plasmids.
- Ti Plasmid: Plants. Plasmid recovered from Agrobacterium. Segment of DNA from plasmid is transfered to plant cell genome. Tumor-causing elements removed from T-DNA.
- Cosmid: Created from plasmid AND virus components.
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Bacteria most common for prok genes
Yeast most common for euk genes
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What is required of a DNA cloning vector?
- Several restriction sites that allow insertion of the DNA fragments to be clones
- intoduced into host cells to allow for independent replication of the DNA fragment
- Selectible marker gene to show they have been taken up
- Easy to isolate from host cell for DNA recovery
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What are some examples of medicinal products produced by recombinant DNA technology?
- Humulin (insulin)
- Erythropoitin
- Human growth hormone
- Hepatitis B vaccine
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What are some examples of genetically modified crops?
- Herbicide-resistance corn/soybeans
- Golden rice
- virus-resistant papaya
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What are some selection methods used to screen for host cells that have taken up the recombinant DNA molecules?
- Blue/white selection: The only bacteria that grow have taken up a plasmid (ampicillin resistance), blue colonies have a working lacZ gene and do not contain the desired DNA, white colonies have a disrupted lacZ gene and contain the desired product.
- Growth on antibiotic medium
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Explain how biotechnology products can be produced by using recombinant DNA technology
- 1) the appropriate vector is determined based on size and type of gene
- 2) the gene and vector are cut using the same restriction enzyme and attempted to be hybridized
- 3) the hybridized vector is inserted into its appropriate host
- 4) the host accepts the vector and vector replicates the gene product
- 5) a selection is made using the selective marker to determine which hosts have the foreign DNA
- 6) these hosts are harvested to retrieve the desired product
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