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Biotechnology

Biotechnology

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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. Synthetic Biology Colonies of the transformed Mycoplasma mycoides bacterium. Image Credit: J. Craig Venter Institute.

  6. 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

  7. 2. Transgenic plants (24.2) Bt-Corn = corn + European corn borer cry gene Bacillus thuringiensis

  8. 3. Transgenic Animals (24.3) GloFish® Assortment gfp gene = jellyfish + Zebra fish

  9. 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

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

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

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

  13. Tools of the Trade

  14. PCR:DNA amplification in vitro • Once you know the sequence of part of a gene, you can amplify it • PolyermaseChain Reaction Kary Mullis winner of the Nobel Prize in chemistry

  15. 11-21

  16. 11-21

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

  18. 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 = Thermusaquaticuswhich is a thermal vent bacteria • Repeat over and over, exponential amplification

  19. Thermusaquaticus Thermusaquaticus Thermal pool in Yellowstone National Park

  20. 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

  21. 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

  22. 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

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

  24. 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

  25. Dideoxy Sequencing Steps

  26. 11-18

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

  28. 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

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

  30. Clone-by-clone strategy 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

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

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

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

  34. 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

  35. 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)

  36. 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.

  37. 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)

  38. 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

  39. Standard kbp 1 2 3 7 6 5 4 3 2 1 Direction of migration Application 2 3. DNA fingerprinting: finding diseased alleles • Person 1 has the disease allele… • Person 2 does NOT have the disease allele • YOU are person 3 • Do you want to know?

  40. Gene Expression - Microarray Thousands of dots per array. Each dot is a single gene Studies transcriptome – sequence and expression patterns of all transcripts

  41. RT=reverse transcriptase Glass slide with 10,000 genes/transcripts

  42. Microarray cluster analysis http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

  43. Next-generation sequencing

  44. Illumina technology

  45. New technology ~ 2.8 Mb of sequence per day ~ 75 Gb of sequence per day ~ 2,700 times more per day!

  46. Data overload

  47. But who cares?