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Biotechnology

Biotechnology. Biotechnology. The use of microorganisms, or biological substances, such as enzymes, to perform specific industrial or manufacturing processes. Science Fact or Fiction?. http://www.flickclip.com/flicks/jurassicpark3.html. Genetic Engineering. The process of producing

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Biotechnology

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  1. Biotechnology

  2. Biotechnology The use of microorganisms, or biological substances, such as enzymes, to perform specific industrial or manufacturing processes

  3. Science Fact or Fiction? http://www.flickclip.com/flicks/jurassicpark3.html

  4. Genetic Engineering The process of producing modified DNA in a test tube and reintroducing that DNA into host organisms.

  5. 1. Gene Therapy (24.5) Gene therapy No gene therapy http://www.vet.upenn.edu/schoolresources/communications/publications/bellwether/55/gene_therapy.html Dogs with the genetic disease mucopolysaccharidosisVII (MPS VII) - enzyme deficiency leads to clouding of corneas, cardiac disease, bone abnormalities - retroviral vector for neonatal gene therapy

  6. 2. Transgenic plants (24.2) Seedless watermelon http://www2.ctahr.hawaii.edu/t-star/watermelon.htm Herbicide resistant plants http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/T/TransgenicPlants.html Silver Gold Rice The biological war on drugs. Herbicide resistant Marijuana??? http://www.washingtontimes.com/news/2006/dec/22/20061222-110251-2050r/

  7. 3. Transgenic Animals (24.3) GloFish® Assortment Enviropig http://www.uoguelph.ca/enviropig/ University of Guelph

  8. 4. Bioremediation Oil-eating bacteria to clean up toxins in the environment http://www.clarkson.edu/news/view.php?id=1095 Microorganisms are eating creosote and chlorinated phenols in Pensacola, FL

  9. 5. Forensics http://www.guardian.co.uk/science/2007/sep/16/sciencenews http://www.king-george.va.us/content6.cfm?cont_uid=2

  10. 6. Pharmaceutical companies http://www.bio.org/speeches/pubs/er/

  11. 7. Research Understanding gene expression, developmental genetics, cancer, aging, genomes, etc. Nearly all labs use some form of biotechnology

  12. PCR:DNA amplification in vitro • Once you know the sequence of part of a gene, you can amplify it • Polyermase Chain Reaction

  13. 11-21

  14. 11-21

  15. PCR video http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html# 11-21

  16. A few more PCR details • To separate DNA – heat to ~94ºC • Cool mixture for primers to anneal – 50-70ºC • Heat mixture to allow Taq polymerase to elongate strands – 68-72ºC • Taq = Thermusaquaticus which is a thermal vent bacteria • Repeat over and over, exponential amplification

  17. PCR Disadvantages Advantages • Amplify a specific sequence • Efficient with very small samples (a few cells) • Great technique for finding homologous genes in other species • You need information in order to design primers • Only amplifies reliably segments less than 2kb

  18. Cloning • ExpressionCloning – isolate/identify a gene of interest to make its protein for medicine, bioremediation, antibodies etc. • Standard Cloning – isolate and identify gene of interest to make lots of it to study for basic research and sequencing • Whole-animalCloning – to generate transgenic animals, to assist infertile couples, and for tissue regeneration and transplant

  19. 1. Recombinant DNA plasmid Restriction enzyme

  20. 2. Getting the recombinant plasmid into bacteria 3. Amplification through cloning (next)

  21. pUC18 vector Ampicillin resistant gene Beta-galactosidase: Works on substrate called X-gal for blue/white colony recognition Bacterial origin of replication Specific information on cloning

  22. Vector not taken by cell One Way…….. Ampicillin resistance Cut foreign DNA and vector with appropriate restriction enzyme Transform into bacteria Bacterial cell Bacterial cell Put onto plate with “food” and ampicillin Collect colonies and sequence Vector in cell Will grow on ampicillin Will not grow on ampicillin

  23. Another Way…….. Blue/White colonies X-gal + working lacZ = blue X-gal + interrupted lacZ = white X-gal cannot be converted into blue color because no working lacZ

  24. Plasmid video • http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

  25. Cloning a phage

  26. Differences between bacterial and phage cloning • Vector into bacteria • Small, singular insert • Ampicillin (or other related gene) resistance • Blue/white colonies • Plate onto Agar “food” and other supplements • Vector into phage • Longer, multiple inserts • Plate onto E. coli and see plaques

  27. Making libraries • One clone represents one part of a genome • cDNA library: a collection of cDNA clones • Genomic library: collection of recombinant DNA-bearing bacteria or phages • Use a shotgun approach to clone a large sample of fragments • “Probes” allow to screen clones for a specific gene

  28. Synthesis of cDNA cDNA video http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html# 11-2

  29. Collection of bacteria with inserts is a library Reverse transcriptase

  30. Probes • Probes: used to identify specific sequences • Probes can be made from: • cDNA from a tissue that expresses a gene of interest at high levels – insulin example • A homologous gene from a related organism • The protein product of the gene of interest • Labeled free RNA • Probes can be radioactive, fluorescent, or chromatic dyes

  31. Radioactive probe searching a phage library

  32. Applications of specific probes • Southern Blotting: detects specific DNA molecule • Dr. Southern developed the technique • Northern Blotting: detects specific RNA molecule • Western Blotting: detects specific protein • Also called DNA and RNA hybridization

  33. Squencing:Determine a specific sequence of DNA • Dideoxy sequencing (Sanger sequencing) • Dideoxy comes from dideoxynucleotidetriphosphate (ddNTP) – blocks DNA synthesis • Lacks the 2’ and 3’-hydroxyl groups (also absent is deoxyribonucleotides)

  34. Dideoxy Sequencing Steps • Denature the 2 strands of DNA • Create a primer for DNA synthesis which will hybridize to exactly one location on the DNA • Add a special “cocktail” of DNA polymerases, normal nucleotide triphosphates, and a small amount of dideoxynucleotides for 1 of the 4 bases • Repeat step #3 with the other 3 dideoxynucleotides

  35. Dideoxy Sequencing Steps

  36. 11-18

  37. Now we use automated sequencing - capillaries • Each ddNTP is tagged with a different fluorescent dye 11-19

  38. Sequencing conclusions • Determines nucleotide sequence of a given strand of DNA • Up to about 1kb or 1000 nucleotides at one time • Now use capillary machines that tell us exact sequence by fluorescent dyes

  39. Whole-Genome Sequencing Race • Shotgun vs. clone-by-clone • Shotgun • J. Craig Venter and The Institution for Genome Research (TIGR) (Private) • Haemophilus influenzae, 1995 • Clone-by-clone • Francis Collins and NIH (Public)

  40. Chromosome Yeast artificial chromosome Different vectors can be used e.g. BACs, plasmids Make overlapping clones and put into smaller vector Clones sequenced then aligned and assembled

  41. Shotgun approach Genomic DNA (or cDNA) 1. Cut DNA 2. Clone fragments 3. Randomly sequence fragments 4. Align sequences 5. Determined sequence

  42. Sequencing Comparison Clone-by-clone Shotgun • Advantages • Fast to get sequences • Cheaper • Disadvantages • Complicated • reassembly (contigs) • Advantages • Easy reassembly • Disadvantages • Slow • Costly

  43. Genetic markers – identifiable DNA sequence differences that distinguish Specific genes OR alleles Specific individuals in a population DNA fingerprinting

  44. Markers • Can be derived from any molecular approach that revels DNA sequence differences • RFLPs – presence/absence of restriction sites • Variable number of tandem repeats (VNTR) • SNPs – rare substitution differences, about 0.001 frequency in humans (1 SNP every 1000bp) • PCR sites – presence/absence of priming sites

  45. 3kbp 4kbp 2kbp 5’ 3’ GAATTC GAATTC CTTAAG 5’ 3’ CTTAAG 3kbp 4kbp 2kbp G 5’ 3’ G AATTC AATTC CTTAA G 5’ 3’ CTTAA RFLPs Restriction sites can be used to identify polymorphisms in the population Person 1 EcoRI • 3 fragments: 2kbp, 3kbp, and 4kbp. • Restriction fragment length polymorphisms (RFLP)

  46. 3kbp 4kbp 2kbp 5’ 3’ GAATTT GAATTC CTTAAA 5’ 3’ CTTAAG 4kbp AATTC 3’ 3kbp 2kbp G 5’ G 5’ G AATTC CTTAA 3’ CTTAA Person 2 EcoRI • 2 fragments: 2kbp, and 7kbp.

  47. Standard kbp 1 2 Slowest, largest RFLPs 7 6 5 4 3 2 1 Direction of migration Fastest, shortest RFLPs • Take these fragments and run them on an agarose or acrylamide gel (electrophoresis) • Ethidium bromide – intercalating dye that binds to DNA and fluorescence under UV light (non specific)

  48. Standard kbp 1 2 3 7 6 5 4 3 2 1 Direction of migration Application 1 • DNA fingerprinting: forensics • E.g. an unknown (person 3), from a crime scene, is added to the known

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