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  1. Chapter 6 Biotechnology: DNA Technology & Genomics 1

  2. The BIG Questions… • How can we use our knowledge of DNA to: • diagnose disease or defect? • cure disease or defect? • change/improve organisms? • What are the techniques & applications of biotechnology? • Biotechnology • The use of living organisms (or substances from living organisms) for practical purposes 2

  3. Biotechnology • Use of organisms to create a desired product is not new • humans have been doing this for thousands of years • Alcohol fermentation (for brewing beer and food preservation) 3

  4. Biotechnology • Agriculture • Plant and animal breeding • Selective breeding – altered the genomes of crops by breeding them with other plants. 4

  5. Biotechnology today • Genetic Engineering • Direct manipulation of DNA • if you are going to engineer DNA & genes & organisms, then you need a set of tools to work with • this chapter is a survey of those tools… 5

  6. Bioengineering Tool kit • Basic Tools • restriction enzymes • ligase • plasmids / cloning • DNA databases / probes • Advanced Tools • PCR • DNA sequencing • gel electrophoresis • Southern blotting • microarrays

  7. Isolating DNA Before DNA can be manipulated, it needs to be isolated from cells. • 1. Cell membranes are disrupted • use a detergent • Salt used to neutralize lipids • 2. DNA precipitation • ethanol used to dehydrate and aggregate DNA • Salt used to neutralize phosphate groups in DNA • 3. DNA isolation / storage

  8. DNA Isolation

  9. 1. Cut DNA • To study the functions of individual genes, molecular biologists will • cut desired genes out of a genome • place the gene into bacterial plasmid • produce recombinant DNA

  10. 1. Cut DNA • treat isolated DNA with restriction enzymes • restriction endonucleases • Evolved in bacteria • Immune System • protection against viruses & other bacteria

  11. Restriction enzymes - Action • cut DNA at specific sequences • Restriction (recognition) site • Are usually 4-8 bp in length • produces protruding ends • sticky ends that contain DNA nucleotides that lack complementary bases • Some do not produce protruding ends • blunt ends CTGAATTCCG GACTTAAGGC  CTG|AATTCCG GACTTAA|GGC  11

  12. Restriction Enzymes • Many different enzymes • named after organism they are found in • EcoRI, HindIII, BamHI, SmaI

  13. Restriction Enzyme Cutting sticky ends – enzyme digests (cuts) to make overhangs EcoRI 5’ G A A T T C 3’ 3’ C T T A A G 5’ 5’ G 3’ 5’ A A T T C 3’ 3’ C T T A A 5’ 3’ G 5’ 5’ overhang

  14. Restriction Enzyme Cutting PstI 5’ C T G C A G 3’ 3’ G A C G T C 5’ 5’ C T G C A 3’ 5’ G 3’ 3’ G 5’ 3’ A C G T C 5’ 3’ overhang

  15. 2. Paste DNA • Sticky ends allow: • H bonds between complementary bases to anneal • DNA Ligase • enzyme “seals” strands • bonds sugar-phosphate backbone together • Condensation reaction 15

  16. DNA Ligase • T4 DNA ligase • originated in T4 bacteriophages • used to chemically join two blunt ends of DNA together

  17. GAATTC GAATTC CTTAAG CTTAAG Biotech use of restriction enzymes DNA Restriction enzyme cuts the DNA Sticky ends (complementary single-stranded DNA tails) AATTC AATTC G G G CTTAA G CTTAA Add DNA from another source cut with same restriction enzyme AATTC G G AATTC CTTAA G DNA ligase joins the strands. 17 GAATTC Recombinant DNA molecule CTTAAG

  18. Exercise 1

  19. Cut, Paste, Copy, Find… • Word processing metaphor… • 1. cut • Isolate desired DNA • restriction enzymes • 2. paste • ligase • 3. copy • plasmids • bacteria • transformation • PCR • 4. find • Southern blotting / probes 19

  20. Remember… • Prokaryotic genomes (e.g. bacteria) contain • Chromosome • Plasmids Chromosome Plasmid

  21. Plasmid pBR322 • Plasmids • Contain “accessory genes” • Antibiotic resistance • Can carry and express foreign genes • Plasmids are vectors • Vehicles by which DNA can be introduced into host cells

  22. Plasmid Restriction Maps • Shows the location of cleavage sites for many different enzymes • These maps are used like road maps to the DNA molecule • Numbers beside restriction enzyme indicates at which base pair the DNA is cut by that particular enzyme

  23. Sample Problem: • Determine the size and number of fragments that would be produced if the plasmid was digested with: • EcoRV and Pvu II? • 2 cuts, therefore 2 fragments • Between sites: 2064-185=1879 bp • Rest: 4361-1879=2482 bp

  24. Cell containing geneof interest Bacterium Gene of interest Plasmid Bacterialchromosome DNA ofchromosome RecombinantDNA (plasmid) Recombinatebacterium 3 Gene of interest Protein expressedby gene of interest Copies of gene Protein harvested Basic research on protein Basic research on gene Gene used to alterbacteria for cleaningup toxic waste Human growth hormone treatsstunted growth Gene for pestresistance inserted into plants Protein dissolvesblood clots in heartattack therapy Why bacteria? • Plasmid uptake is easy • Cheap • Reproduce rapidly and frequently • Produce multiple copies of the recombinant DNA and protein in a short amount of time • Recombinant DNA and proteins are kept in large storage database to be used later

  25. Protein Databases Actin Protein Unnamed Protein

  26. Review: Transformation vs. Recombination • Transformation • the introduction of foreign DNA (usually a plasmid) into a bacterial cell • Recombination • Fragment of DNA composed of sequences originating from at least two different sources

  27. 3. Copying (Cloning) 1 2 3 In this example, a human gene is inserted into a plasmid from E. coli. The plasmid contains the ampR gene, which makes E. coli cells resistant to the antibiotic ampicillin. It also contains the lacZ gene, which encodes -galactosidase. This enzyme hydrolyzes a molecular mimic of lactose (X-gal) to form a blue product. Only three plasmids and three human DNA fragments are shown, but millions of copies of the plasmid and a mixture of millions of different human DNA fragments would be present in the samples. TECHNIQUE lacZ gene (lactose breakdown) Bacterial cell Isolate plasmid DNA and human DNA. Human cell Restriction site Cut both DNA samples with the same restriction enzyme ampR gene (ampicillin resistance) Bacterial plasmid Gene of interest Stickyends Human DNAfragments Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Figure 20.4 Recombinant DNA plasmids

  28. 4 5 RESULTS Introduce the DNA into bacterial cells (transoformation). Recombinantbacteria Colony carrying non-recombinant plasmid with intact lacZ gene Colony carryingrecombinant plasmidwith disrupted lacZ gene Plate the bacteria on agar containing ampicillin and X-gal (lactose). Incubate until colonies grow. Only a cell that took up a plasmid, which has the ampR gene, will reproduce and form a colony. Colonies with nonrecombinant plasmids will be blue, because they can hydrolyze X-gal. Colonies with recombinant plasmids, in which lacZ is disrupted, will be white, because they cannot hydrolyze X-gal. By screening the white colonies with a nucleic acid probe (see Figure 20.5), researchers can identify clones of bacterial cells carrying the gene of interest. Bacterialclone

  29. How do we know if it worked? • We know cloning worked if: • Transformation has occurred • i.e. there has been plasmid uptake into the bacteria • Recombination has occurred • i.e. foreign genes have been inserted into the bacterial plasmid

  30. Selecting for successful transformation • Antibiotic resistance genes as a selectable marker • Plasmid has both “added” gene & antibiotic resistance gene selection 30

  31. Amp Selection • If bacteria don’t pick up plasmid • they will not have antibiotic resistance • die on antibiotic (amp) plates • If bacteria pick up plasmid • survive on antibiotic (amp) plates • We have selected only those colonies that have undergone successful transformation

  32. Amp Selection • Ampicillin is the selecting agent only transformed bacteria grow all bacteria grow LB plate LB/amp plate 32

  33. Screening for successful recombination • Transformed colonies • have both recombinant and non-recombinant plasmids recombinant non-recombinant

  34. restriction sites insertedgeneof interest LacZ gene brokenLacZ gene X lactose  white color lactose  blue color recombinantplasmid plasmid amp resistance amp resistance origin ofreplication LacZ Screening LacZ gene codes for β galactosidase Lactose (or X-gal) is our screening agent EcoRI all in LacZ gene BamHI HindIII 34

  35. LacZ screening system • Make sure inserted plasmid is recombinant plasmid • LacZ gene on plasmid produces digestive enzyme • lactose(X-gal)  blue • blue colonies • insert foreign DNA into LacZ gene breaks gene • lactose (X-gal)  blue • white colonies • white bacterial colonies have recombinantplasmid X X We wantthese!! 35

  36. Amp selection & LacZ screening • gene of interest • LacZ gene • - amp resistance LB/amp LB/amp/Xgal 36

  37. Cut, Paste, Copy, Find… • Word processing metaphor… • cut • restriction enzymes • paste • ligase • copy • plasmids • bacteria • transformation • PCR • find • Southern blotting / probes     37

  38. 4. Find • White colonies could have recombinant plasmids • desired and undesired genes • How do you find the conony with the gene of interest?

  39. But howdo we findcolony with our gene of interestin it? 4. Find recombinant plasmids inserted into bacteria gene of interest bacterial colonies (clones) grown on LB/amp/Xgal petri plates 39

  40. labeled probe G A T C A G T A G genomic DNA C T A G T C A T C 5’ 3’ Locating your gene of interest • DNA hybridization • find gene in bacterial colony using a probe • short, single stranded DNA molecule • complementary to part of gene of interest • tagged with radioactive P32 or fluorescence • heat treat genomic DNA • unwinds (denatures) strands • DNA hybridization between probe & denatured DNA

  41. Hybridization 4 • Locate • expose film • locate colony on plate from film Cloning - plate with bacterial colonies carrying recombinant plasmids 1 plate plate + filter film 2 • Replicate plate • press filter paper onto plate to take sample of cells from every colony Hybridization - heat filter paper to denature DNA - wash filter paper with radioactive probe which will only attach to gene of interest filter 3

  42. Classwork/Homework • Pg. 281 #1-3 • Pg. 282 #6,7,10 • Pg. 287 #16,18 • Pg. 289 #20,21 • Pg. 291 #2-7,10,11(b,c),15,16(a,b),17,19 • Pg. 295 #4,5