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A Lot More Advanced Biotechnology Tools

A Lot More Advanced Biotechnology Tools. DNA Sequencing. DNA Sequencing. dideoxynucleotides ddATP, ddGTP, ddTTP, ddCTP missing O for bonding of next nucleotide terminates the growing chain. 2. 1. 3. 4. 2. DNA Sequencing. Sanger method synthesize complementary DNA strand in vitro

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A Lot More Advanced Biotechnology Tools

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  1. A Lot More Advanced Biotechnology Tools • DNA Sequencing

  2. DNA Sequencing • dideoxynucleotides • ddATP, ddGTP, ddTTP, ddCTP • missing O for bonding of nextnucleotide • terminates the growing chain

  3. 2 1 3 4 2 DNA Sequencing • Sanger method • synthesize complementary DNA strand in vitro • in each tube: • “normal” N-bases • dideoxy N-bases • ddA, ddC, ddG, ddT • DNA polymerase • primer • buffers & salt

  4. Reading the sequence • Load gel with sequences from ddA, ddT, ddC, ddG in separate lanes • read lanes manually & carefully • polyacrylamide gel

  5. Fred Sanger 1978 | 1980 • This was his 2nd Nobel Prize!! • 1st was in 1958 for the structure of insulin

  6. The Sanger Method: DNAi video tutorial

  7. Advancements in sequencing • Fluorescent tagging • no more radioactivity • all 4 bases in 1 lane • each base a different color • Automated reading

  8. More Advancements in sequencing • Capillary tube electrophoresis • no more pouring gels • higher capacity & faster Applied Biosystems, Inc (ABI) built an industry on these machines

  9. Big labs! • economy of scale • PUBLIC • Joint Genome Institute (DOE) • MIT • Washington University of St. Louis • Baylor College of Medicine • Sanger Center (UK) • PRIVATE • Celera Genomics

  10. Automated Sequencing machines • Really BIG labs!

  11. Human Genome Project • U.S government project • begun in 1990 • estimated to be a 15 year project • DOE & NIH • initiated by Jim Watson • led by Francis Collins • goal was to sequence entire human genome • 3 billion base pairs • Celera Genomics • Craig Venter challenged gov’t • would do it faster, cheaper • private company

  12. Different approaches Craig Venter’s method gov’t method “map-based method” “shotgun method” 1. Cut DNA entire chromosome into small fragments and clone. 2. Sequence each segment & arrange based on overlapping nucleotide sequences. • Cut DNA segment into fragments, arrange based on overlapping nucleotide sequences, and clone fragments. • 2. Cut and clone into smaller fragments. 3. Assemble DNA sequence using overlapping sequences.

  13. Shotgun!

  14. Human Genome Project • On June 26, 2001, HGP published the “working draft” of the DNA sequence of the human genome (4 years ahead of schedule). • Historic Event! • blueprint of a human • the potential to change science & medicine

  15. Sequence of 46 Human Chromosomes 3G of data 3 billion base pairs

  16. Things Are Strange In Here:

  17. Raw genome data

  18. NCBI GenBank • Database of genetic sequences gathered from research • Publicly available on Web!

  19. Maps of human genes… • Where the genes are… • mapping genes & their mutant alleles

  20. gene gene polypeptide 1 Defining a gene… protein RNA gene polypeptide 2 polypeptide 3 • “Defining a gene is problematic because… one gene can code for several protein products, some genes code only for RNA, two genes can overlap, and there are many other complications.” • – Elizabeth Pennisi, Science 2003

  21. Exons (regions of genes coding for protein, rRNA, tRNA) (1.5%) Types of DNA sequences in the human genome Repetitive DNA that includes transposable elements and related sequences (44%) Introns and regulatory sequences (24%) Unique noncoding DNA (15%) Repetitive DNA unrelated to transposable elements (about 15%) Alu elements (10%) Simple sequence DNA (3%) Large-segment duplications (5–6%)

  22. And we didn’t stop there…

  23. Genome Sizes and Estimated Numbers of Genes*

  24. What have we found? • When you go looking…

  25. …you will certainly find something!

  26. Sing A Silly Song Extolling Automation!

  27. Ethical Questions…

  28. 1. You have a familiar history of a terminal genetic disease. A genetic test exists for the disease. Would you take the test to see if you will have the disease?

  29. 2. As a condition of your continued employment, your boss wants you to have a genetic screening test and the results sent to the company. Will you get the test?

  30. You and your spouse have decided to have a child. You have the resources to pre-determine your child’s gender. Would you do this? • What about your child’s intelligence?

  31. 5. You discover your favorite food contains genetically modified ingredients. Will you continue to eat the food?

  32. As part of a routine medical procedure, your doctor discovers that you have a rare, beneficial variant of a protein that protects you from heart disease. Should your doctor be able to patent the protein? • Should you be entitled to any money from the patent?

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