Biotechnology Chapter 20

1. BiotechnologyChapter 20

2. DNA technology • Sequencing & manipulation of DNA • Used in analyzing gene expression

3. DNA sequencing

4. Biotechnology • Manipulation of organisms to make useful products

5. DNA Sequencing • Nucleic acid hybridization: • Complementary base pairing • One stand onto a different strand • 2 techniques • Dideoxy chain termination • Next-generation sequencing

6. Technique Dideoxyribonucleotides(fluorescently tagged) DNA (template strand) Primer Deoxyribo-nucleotides 3′ T G T T 5′ C T G AC T T C G A C A A dATP ddATP Dideoxy chain termination 5′ dCTP ddCTP DNA polymerase dTTP ddTTP dGTP ddGTP P P P P P P G G 3′ Labeled strands DNA (template strand) 5′ 3′ dd G A C T G A C T G AC T T C G A C A A dd A C T G A A G dd C T G A A G dd T G A A G dd G A A G dd A A G dd A G A G C T G T T dd G C T G T T 3′ dd C T G T T C T G T T C T G T T C T G T T C T G T T C T G T T C T G T T 5′ 5′ 3′ Shortest Longest Directionof movementof strands Longest labeled strand Detector Laser Shortest labeled strand Results Last nucleotideof longestlabeled strand G A C T G A A G C Last nucleotideof shortestlabeled strand

7. Technique Genomic DNA is fragmented. Results Next generation A 4-mer T Each fragment is isolated witha bead. G 3-mer C 2-mer Using PCR, 106 copies of eachfragment are made, each attachedto the bead by 5′ end. 1-mer The bead is placed into a well withDNA polymerases and primers. Template strandof DNA 3′ 5′ 3′ 5′ G A T C Primer A solution of each of the four nucleotidesis added to all wells and then washed off.The entire process is then repeated. G G G A T C A T C A T C A T C G Templatestrandof DNA C C C C C C C C dCTP A A A A dTTP dGTP A A A A dATP T T T T C G G G G PPi A A A A T T T T PPi C C C C G G G G DNApolymerase C C C C G G G G A A A A G G G G Primer T T T T A A A A 8 4 5 3 6 1 2 If a nucleotide is notcomplementary to thenext template base,no PPi is released, andno flash of light is recorded. The process is repeated until everyfragment has a complete complementarystrand. The pattern of flashes reveals thesequence. If a nucleotide is joined to a growing strand, PPi is released, causing a flash of light that is recorded. 7

8. Genetic engineering • Manipulation of genes • Gene cloning: • Multiple copies of a single gene • Produce a specific product

9. Fig. 20-2 Cell containing geneof interest Bacterium 1 Gene inserted intoplasmid Bacterialchromosome Plasmid Gene ofinterest RecombinantDNA (plasmid) DNA of chromosome 2 Plasmid put intobacterial cell Recombinantbacterium 3 Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest Gene ofInterest Protein expressedby gene of interest Copies of gene Protein harvested Basic research andvarious applications 4 Basicresearchon protein Basicresearchon gene Gene used to alter bacteria for cleaning up toxic waste Gene for pest resistance inserted into plants Protein dissolvesblood clots in heartattack therapy Human growth hor-mone treats stuntedgrowth

10. Recombinant DNA • 1970’s • Combining genes from different sources • Even different species • Combined into single DNA • Example: Bacteria & mammal

11. Recombinant DNA • Genetically modified bacteria • Mass produce beneficial chemicals • Insulin • Growth hormone • Cancer drugs • Pesticides

12. Plasmid

13. Plasmid • Small separate circular DNA • Replicated same as main DNA • Foreign DNA added to plasmid • Replicated along with plasmid

14. Recombinant DNA • Nucleases: • Enzymes that degrade DNA • Restriction endonulceases: • Restriction enzymes • Cut DNA into fragments • Specific points

15. Recombinant DNA • Restriction sites: • Where DNA is cut • Restriction fragments • Short DNA sequence

16. Recombinant DNA • “sticky ends” • Cuts in DNA sequences • Single-stranded ends

17. Figure 20.6a Bacterialplasmid Restriction site 1 5′ 3′ GAATTC DNA CTTAAG 3′ 5′ Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow. 5′ 3′ 3′ 5′ AATTC G CTTAA G 5′ 3′ 5′ 3′ Sticky end

18. Sticky ends

19. Recombinant DNA • Insertion of DNA fragments from other sources • Sticky ends • Complementary match base pairs • Hydrogen bonds • DNA ligase: • Forms a phosphodiester bond

25. Recombinant DNA (Process) • 1. Isolate gene of interest & bacterial plasmid • 2. Cut DNA & plasmid into fragments • 3. Mix DNA fragments with cut plasmid. • Fragment with gene of interest is inserted into the plasmid • 4. Recombinant plasmid is mixed with bacteria

26. Recombinant DNA (Process) • 5. Bacteria with recombinant DNA reproduce • 6. Isolate bacterial clones that contain gene of interest • Producing protein of interest • 7. Grow large quantities of bacteria that produce the protein

27. Recombinant DNA (Process)

28. Hummingbird cell TECHNIQUE Fig. 20-4-4 Bacterial cell lacZ gene Restrictionsite Stickyends Gene of interest Bacterial plasmid ampR gene Hummingbird DNA fragments Nonrecombinant plasmid Recombinant plasmids Bacteria carryingplasmids RESULTS Colony carrying recombinant plasmid with disrupted lacZ gene Colony carrying non-recombinant plasmidwith intact lacZ gene One of manybacterial clones

29. Recombinant DNA • Vector: • DNA molecule-carries foreign DNA • Enters & replicates in the host • Plasmids & phages are common vectors • Phages are larger than plasmid • Can handle inserts up to 40 kilobases

30. PCR • Polymerase chain reaction • Amplify DNA • Makes large quantities of DNA • 1985

31. PCR • Heated • Denatured • DNA primer • Heat stable DNA polymerase • Makes DNA

32. Fig. 20-8 3 5 TECHNIQUE Targetsequence 3 5 Genomic DNA 1 5 3 Denaturation 5 3 2 Annealing Cycle 1yields 2 molecules Primers 3 Extension Newnucleo-tides Cycle 2yields 4 molecules Cycle 3yields 8 molecules;2 molecules(in whiteboxes)match targetsequence

33. Gel electrophoresis • Study DNA • Polymer (gel) • Restriction fragments • Separates DNA based on charge & size • Nucleic acids negative charge (Phosphates) • Migrate towards + end (red)

34. Fig. 20-9 TECHNIQUE Powersource Mixture ofDNA mol-ecules ofdifferentsizes – Cathode Anode + Gel 1 Powersource – + Longermolecules 2 Shortermolecules RESULTS

35. Fig. 20-10 Normal -globin allele Normalallele Sickle-cellallele 175 bp Large fragment 201 bp DdeI DdeI DdeI DdeI Largefragment Sickle-cell mutant -globin allele 376 bp 201 bp175 bp Large fragment 376 bp DdeI DdeI DdeI (a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles

36. Analyze gene expression • cDNA • Complementary DNA • DNA made from an mRNA • mRNA where gene is expressed • RT-PCR • Reverse transcriptase polymerase chain reaction

37. Analyze gene expression • In vitro mutagenesis • Cloned mutated gene • Blocks expression • RNAi • RNA interference • Nematodes, fruit fly

38. Analyze gene expression • Genetic markers • Detect abnormal disease • SNP • Single nucleotide polymorphisms • Single base pair site where variation is found • RFLP • Restriction fragment length polymorphisms

39. Fig. 20-21 DNA T Normal allele SNP C Disease-causingallele

40. Cloning • Multicellular organisms come from a single cell. • Offspring are identical

41. Cloning • 1950 • Carrots • Totipotent: • Mature cells-undifferentiated • Give rise to any type of cells • Common in plants

42. Cloning • Nuclear transplantation • Nucleus of an unfertilized/fertilized egg is removed • Replaced with nucleus of differentiated cell • Direct development of cell into tissues etc.

43. Cloning • Removed nuclei from an egg • Mammary cells • Fused with egg cells • Dolly, 1997, identical to mammary cell donor • Died prematurely age 6 • Arthritis & lung disease

44. TECHNIQUE Fig. 20-18 Mammarycell donor Egg celldonor 2 1 Egg cellfrom ovary Nucleusremoved Cells fused 3 Culturedmammary cells 3 Nucleus frommammary cell Grown inculture 4 Early embryo Implantedin uterusof a thirdsheep 5 Surrogatemother Embryonicdevelopment 6 Lamb (“Dolly”)genetically identical tomammary cell donor RESULTS

46. Fig. 20-19

47. Cloning • Few develop normally • Abnormalities • Epigenetic changes to the chromatin • More methylation of chromatin • Reprogram chromatin of differentiated cell

48. Stem cells • Started 1998 at UW • Early embryonic cells • Potential to become any type of cell • Master cell generates specialized cells • Such as muscle cells, bone cells, or blood cells

49. Stem cells • Embryos • Bone marrow • Umbilical cord blood • Blood stem cells • iPS • Induced pluripotent stem cells • Skin cells

50. Embryonic stem cells Adult stem cells From bone marrowin this example Early human embryoat blastocyst stage(mammalian equiva-lent of blastula) Fig. 20-20 Cells generatingall embryoniccell types Cells generatingsome cell types Culturedstem cells Differentcultureconditions Differenttypes ofdifferentiatedcells Blood cells Nerve cells Liver cells