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Biotechnology

Biotechnology. Biotechnology - the use of microbes or cells to make a product.Historical Perspective- where did this revolution come from?Years of Government investing $$ into long-term scientific research via agencies like the NIH, CDC, and NSF.Since 1970, every $1 invested has yielded over $10

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Biotechnology

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    2. Biotechnology Biotechnology - the use of microbes or cells to make a product. Historical Perspective- where did this revolution come from? Years of Government investing $$ into long-term scientific research via agencies like the NIH, CDC, and NSF. Since 1970, every $1 invested has yielded over $10 in economic stimulus. Companies invest some but not like the Govt.; tends to be short-term.

    3. Biotechnology Recombinant DNA - genes taken from one organisms and inserted into another. Insulin gene from humans into E. coli. Cloning - making new strains.

    4. Genetic Engineering Need a vector such as a plasmid to shuttle gene. Gene is isolated from donor organism.

    5. Genetic Engineering 3. Gene is inserted into the plasmid. 4. Plasmid is inserted into recipient bacterium.

    6. Genetic Engineering 5. New clone grown. 6. Either copies of the gene are made or the product is induced.

    7. Genetic Engineering Use restriction endonucleases to cut DNA. Each enzyme cuts a specific pattern of bases. There are hundreds of restriction endonucleases .

    8. Restriction Endonucleases Where did they come from? They are DNA housekeeping enzymes used to prevent the contamination of a species chromosome with “foreign DNA”. Each species has it’s own set. This is why conjugation and transduction must be between closely related species. Examples: HindIII, BamH1, EcoR1, HpaI

    9. Restriction Endonucleases 3 restriction endonucleases Patterns different 3 different organisms 1 straight cut- blunt ends 2 make “sticky ends”

    10. Genetic Engineering Cut recipient DNA with the same restriction endonuclease. “Sticky ends” will match and be complementary. Use DNA ligase to rejoin segments.

    11. Genetic Engineering One of most common plasmid shuttles is pUC19 (puck -19). Use the antibiotic ampicillin to screen for clones. Sites on the right are Restriction Enzyme locations.

    12. DNA Isolation- Purification How do you isolate DNA? Kits (for almost everything) Lysis buffer to break open cells. Run lysate through column

    13. DNA Isolation- Purification How do you isolate DNA? Wash - remove impurities. Elute.

    14. Selecting a Clone Once you get gene into plasmid and the plasmid into recipient bacteria (E. coli), you need to have a method to select those cells which have accepted the plasmid. Antibiotic resistance is one means; ampicillin, kanamycin, etc

    15. Selecting a Clone In addition, metabolic genes allow you to distinguish between those that pick up the plasmid with the insert and those that pick up the unaltered plasmid. Those with inserts get the altered plasmid and cannot digest X-gal; those with native (unaltered) plasmid get galactose gene and can hydrolyse X-gal (blue).

    16. Applications Products of Genetic Engineering Plasminogen activator Insulin Erythropoietin Interferon EGF HGH HepB vaccine

    17. DNA Sequencing Done with instruments Sequence thousands of base pairs per day. Output in graphs and text documents.

    18. DNA Sequencing Print out shows peaks Color corresponds to base : A =red, T= green, G = black; C = blue; N =red

    19. Southern Blotting Use DNA to bind to sample DNA to probe for defect. Digest sample DNA. Separate by gel electrophoresis.

    20. Southern Blotting Transfer DNA to nitrocellulose (blotting). Electrical field commonly used to push DNA out of gel onto nitrocellulose film.

    21. Southern Blotting Probe blot with radiolabeled DNA segment. Expose X-ray film and develop. Because of hazards of radioactive chemicals, many now use fluorescent compounds.

    22. AI Instrument AlphaImmogen Gel Imager PC driven Excite gel with UV or visible light 1 Mpixel camera digitalizes image. Software to labe and compare gels.

    23. AI Instrument AlphaInnogen Gel Imager PC driven Excite gel with UV or visible light 1 Mpixel camera digitalizes image. Software to labe and compare gels.

    24. AI Instrument AlphaInnogen Gel Imager PC driven Excite gel with UV or visible light 1 Mpixel camera digitalizes image. Software to label, store, and compare gel images.

    25. DNA Fingerprinting Digest 2 samples of DNA with one or more REs. Separate fragments by gel electrophoresis. Compare samples. E. coli from patients and juice samples. Same can be done with human DNA samples for forensic exams.

    26. RT-PCR Used to study Gene Expression. Use reverse transcriptase to make DNA from mRNA.

    27. RT-PCR Lane 1,8 MWt Stds Lane 3 b-actin Lane 4,5 CAP-18 Lane 6 no RT Lane 7 no RNA

    28. Real Time - PCR Same as PCR but you add a probe with a fluorescent label. The probe sequence is between the 2 primers. As DNA polymerase makes copies it chews up probe releasing fluorescent compound. Ideal when results needed ASAP.

    29. Real Time - PCR Signal slowly increases each cycle. Eventually breaks a threshold value See results as a curve. 1.5 - 2 hrs total. Do NOT have to run on a gel. 100 -1000 times more sensitive. Can detect as little as 10 copies of DNA.

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