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The DNA Toolbox

The DNA Toolbox. The Human Genome Project. Genome - all of an organism’s DNA Mapped 3.2 billion base pairs Between 20,000 and 30,00 genes Completed in 2003. A = T C = G. Biotechnology. Manipulation of organisms or their components to make useful products

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The DNA Toolbox

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  1. The DNA Toolbox

  2. The Human Genome Project • Genome- all of an organism’s DNA • Mapped 3.2 billion base pairs • Between 20,000 and 30,00 genes • Completed in 2003 A = T C = G

  3. Biotechnology • Manipulation of organisms or their components to make useful products • Historically, it includes selective breeding of farm animals and crops, and using microorganisms to make wine and cheese • Today, it also includes genetic engineering

  4. Genetic Engineering • Direct manipulation of the DNA molecule itself • Utilizes recombinant DNA

  5. Recombinant DNA • DNA molecules formed when segments of DNA from two different sources (often different species) are combined in vitro (in a test tube)

  6. Genetic engineering requires a special set of tools • This unit is a survey of those tools…

  7. Review • Genes occupy only a small proportion of a chromosome • The coding sequences are called exons • The rest of the chromosome is made up of non-coding segments called introns intron exon gene

  8. DNA Cloning • Scientists needed a way to work directly with desired genes and not the “trash” • DNA cloning is the process by which multiple identical copies of specific DNA is produced

  9. E coli • Most cloning methods involve bacteria • Most commonly used bacteria is E coli

  10. Bacteria Review • Unicellular prokaryotes • Reproduce by binary fission • Short generation span • New offspring every +/- 20 minutes • Colony of 100 million overnight! • Incredibly diverse • Dominant form of life on Earth

  11. Bacterial Genome • Single, circular chromosome • Reproduce asexually so always haploid (n) • Naked DNA (no histones) • ~4 million base pairs; 4300 genes • 1/1000 DNA in eukaryote • Contain plasmids

  12. Plasmids • Small supplemental circles of DNA • Replicate separately from bacterial chromosome • Carry 2-30 genes, including those for antibiotic resistance • Can be imported from environment

  13. transformedbacteria gene fromother organism recombinantplasmid cut DNA vector plasmid Inserting Genes into Bacteria Using Plasmids • Cut plasmid DNA and DNA containing desired gene • Insert new gene into plasmid • Insert recombined plasmid into bacteria + glue DNA

  14. How do we cut DNA? • Restriction enzymes • Evolved in bacteria to cut up foreign DNA • Protect bacteria against invaders • Bacteriophages- viruses that attack bacteria 

  15. What keeps the restriction enzyme from cutting up the bacteria’s own DNA? • Methylation • By using enzymes that DO NOT recognize any of their own base sequences

  16. What do you notice about these phrases? radar racecar Madam I’m Adam a man, a plan, a canal, Panama Was it a bar or a bat I saw? palindromes

  17. Restriction Enzymes Target Palindromes • Which of the following DNA sequences is a palindrome? A. B. C. 5' AAGG 3' 5' AGTC 3' 5' GGCC 3' 3' TTCC 5' 3' TCAG 5' 3' CCGG 5'

  18. Inserting Foreign DNA into a Plasmid • Restriction enzyme reads DNA from 5' to 3' looking for specific sequence: GAATTC • Cuts DNA at restriction site: between G and A • Most useful produce asymetrical cuts resulting in protuding ends called sticky ends • Sticky ends will bind to any complementary DNA  AATTCCG 5' 3' CTGAATTCCG GACTTAAGGC GGC 5' 3' CTG GACTTAA 

  19. Many Different Restriction Enzymes • Discovered in 1960s • Key to genetic engineering • Named after organism in which they were found • E coli Restriction Enzyme I (EcoRI): first to be discovered in E coli • Each one recognizes a unique base sequence so they cut in different places

  20. Common Restriction Enzymes

  21. gene you want chromosome want to add gene to GGACCTG AATTCCGGATA CCTGGACTTAA GGCCTAT GTAACG AATTCACGCTT CATTGCTTAA GTGCGAA GGACCTG AATTCACGCTT CCTGGACTTAA GTGCGAA combinedDNA Sticky ends • Cut other DNA with same enzymes • leave “sticky ends” on both • can glue DNA together at “sticky ends” • DNA Ligase- enzyme that glues sticky ends together

  22. The code is universal • Since all living organisms… • use the same DNA • use the same code book • read their genes the same way

  23. Plasmids and Genetic Engineering cut DNA using restriction enzyme gene we want like what? …insulin …HGH cut plasmid DNA using same enzyme  recombinant plasmid capable of producing desired protein insert “gene we want” into plasmid...“glue” together using DNA Ligase

  24. Insert recombined plasmid to form transformed bacteria Harvest & purifyprotein Transformation Grow bacteria allowing it to produce protein as directed by new gene

  25. ANY QUESTIONS?

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