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Chapter 20

Chapter 20. Biotechnology. Overview: The DNA Toolbox. Sequencing of the human genome was completed by 2007 DNA sequencing has depended on advances in technology, starting with making recombinant DNA

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Chapter 20

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  1. Chapter 20 Biotechnology

  2. Overview: The DNA Toolbox • Sequencing of the human genome was completed by 2007 • DNA sequencing has depended on advances in technology, starting with making recombinant DNA • In recombinant DNA, nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule

  3. Methods for making recombinant DNA are central to genetic engineering (direct manipulation of genes for practical purposes) • DNA technology has revolutionized biotechnology, the manipulation of organisms or their genetic components to make useful products • An example of DNA technology is the microarray, a measurement of gene expression of thousands of different genes

  4. Concept 20.1: DNA cloning yields multiple copies of a gene or other DNA segment • To work directly with specific genes, scientists prepare gene-sized pieces of DNA in identical copies, a process called DNA cloning

  5. I. DNA Cloning • Use bacteria and their plasmids • Remember: Plasmids = small circular DNA molecules that replicate separately from the bacterial chromosome • Cloning is used for making copies of gene and producing a protein product

  6. Gene cloning = uses bacteria to make multiple copies of a gene • Foreign DNA is inserted into a plasmid, and the recombinant plasmid is inserted into a bacterial cell • Reproduction of bacterial cell results in cloning of the recombinant plasmid • Results = production of multiple copies of gene

  7. Fig. 20-2a • Cell containing geneof interest • Bacterium • 1 • Gene inserted intoplasmid • Bacterialchromosome • Plasmid • Gene ofinterest • RecombinantDNA (plasmid) • DNA of chromosome • 2 • 2 • Plasmid put intobacterial cell • Recombinantbacterium

  8. Fig. 20-2b • Recombinantbacterium • Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest • 3 • Protein expressedby gene of interest • Gene ofInterest • Copies of gene • Protein harvested • Basic research andvarious applications • 4 • Basicresearchon protein • Basicresearchon gene • Protein dissolvesblood clots in heartattack therapy • Human growth hor-mone treats stuntedgrowth • Gene for pest resistance inserted into plants • Gene used to alter bacteria for cleaning up toxic waste

  9. II. Restriction Enzymes • Bacterial restriction enzymes (protects bacteria from phages) cut DNA molecules at specific DNA sequences (restriction sites) • Restriction enz makes many cuts, yielding restriction fragments • Rest enz cut DNA in a staggered way, makes fragments with “sticky ends”, that bond with complementary sticky ends of other fragments • DNA ligase = seals bonds between fragments

  10. Fig. 20-3-3 • Restriction site • 5 • 3 • 3 • 5 • DNA • Restriction enzymecuts sugar-phosphatebackbones. • 1 • Sticky end • DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs. • 2 • One possible combination • DNA ligaseseals strands. • 3 • Recombinant DNA molecule

  11. III. Cloning a Euk Gene in a Bacterial Plasmid • cloning vector = the original plasmid that can carry foreign DNA into a host cell and replicate there

  12. A. Producing Cells w/ Recombinant Plasmids • Steps to clone the hummingbird β-globin gene in a bacterial plasmid: • Isolate genomic DNA and a bacterial plasmid • Digest both with SAME rest enz • Fragments are mixed, DNA ligase is added to bond the fragment sticky ends

  13. Some recombinant plasmids now contain hummingbird DNA • DNA mixture is added to bacteria • Bacteria are plated on agar that selects for the bacteria with recombinant plasmids • Results in the cloning of many hummingbird DNA fragments, including the β-globin gene

  14. Fig. 20-4-4 • Hummingbird cell • TECHNIQUE • 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

  15. B. Screening Clones for a Gene • Nucleic acid probe = identifies clone by using a sequence complementary to the gene • This process is called nucleic acid hybridization • For example, if the desired gene is – Then we would synthesize this probe … … 5 G G C T A A C T T A G C 3 C C G A T T G A A T C G 5 3

  16. DNA probe is used to screen a large number of clones at same time for gene of interest • Once identified, the clone can be cultured

  17. Fig. 20-7 • • • TECHNIQUE • Radioactivelylabeled probemolecules • ProbeDNA • Gene ofinterest • Multiwell platesholding library • clones • Single-strandedDNA from cell • Film • Nylon membrane • Nylonmembrane • Location ofDNA with thecomplementarysequence

  18. IV. Expressing Cloned Euk Genes • Protein products of cloned genes can be produced in larger amounts for research • Cloned genes can be expressed in either bacterial or eukaryotic cells

  19. A. Bacterial Expression • Scientists use an expression vector (a cloning vector that contains a highly active prokaryotic promoter) to overcome differences in promoters and other DNA control sequences

  20. B. Eukaryotic Expression • Using eukaryotic cells as hosts and yeast artificial chromosomes (YACs) as vectors helps avoid gene expression problems • YACs can carry more DNA than a plasmid • Euk hosts can provide the post-translational modifications that many proteins require

  21. Electroporation = applying an electrical pulse to create temp holes in plasma membranes introducing recombinant DNA • Can inject DNA into cells using microscopically thin needles

  22. V. Amplifying DNA in Vitro • Polymerase chain reaction (PCR) can produce many copies of a specific target segment of DNA • A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules

  23. 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

  24. DNA Cloning - Andersen

  25. Concept 20.2: DNA technology allows us to study the sequence, expression, and function of a gene • DNA cloning allows researchers to • Compare genes and alleles between individuals • Locate gene expression in a body • Determine the role of a gene in an organism • Several techniques are used to analyze the DNA of genes

  26. VI. Gel Electrophoresis and Southern Blotting • Gel electrophoresis = method of rapidly analyzing and comparing genomes • Uses gel to separate nucleic acids or proteins by size • Electrical current is applied that causes charged molecules to move through the gel • Molecules form “bands” by their size

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

  28. Restriction fragment analysis = DNA fragments from rest enz digestion of DNA are sorted by gel electrophoresis • Used for comparing two different DNA molecules • Used to prepare pure samples of individual fragments

  29. 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

  30. Gel Electrophoresis Virtual Lab

  31. Southern blotting combines gel electro with nucleic acid hybridization • DNA fragments can be identified using labeled probes that bind to the DNA that is stuck on a “blot” of gel

  32. Fig. 20-11a • TECHNIQUE • Heavyweight • Restrictionfragments • I II III • DNA + restriction enzyme • Nitrocellulosemembrane (blot) • Gel • Sponge • I Normal-globinallele • II Sickle-cellallele • III Heterozygote • Papertowels • Alkalinesolution • 2 • 3 • 1 • Preparation of restriction fragments • Gel electrophoresis • DNA transfer (blotting)

  33. Fig. 20-11b • Radioactively labeledprobe for -globin gene • Probe base-pairswith fragments • I II III • I II III • Fragment fromsickle-cell-globin allele • Film overblot • Fragment fromnormal -globin allele • Nitrocellulose blot • 5 • 4 • Hybridization with radioactive probe • Probe detection

  34. VII. DNA Sequencing • Modified nucleotides called dideoxyribonucleotides (ddNTP) attach to synthesized DNA strands of different lengths • Each type of ddNTP is tagged with a distinct fluorescent label that identifies the nucleotide at the end of each DNA fragment • The DNA sequence can be read from the resulting spectrogram • Read from bottom to top (smallest fragment on bottom, this is 5’ end, original DNA is complimentary)

  35. Fig. 20-12a • TECHNIQUE • DNA(template strand) • Primer • Deoxyribonucleotides • Dideoxyribonucleotides(fluorescently tagged) • dATP • ddATP • dCTP • ddCTP • dTTP • ddTTP • DNA polymerase • dGTP • ddGTP

  36. Fig. 20-12b • TECHNIQUE • DNA (template strand) • Labeled strands • Shortest • Longest • Directionof movementof strands • Longest labeled strand • Detector • Laser • Shortest labeled strand • RESULTS • Last baseof longestlabeledstrand • Last baseof shortestlabeledstrand

  37. VIII. Analyzing Gene Expression • Nucleic acid probes can hybridize with mRNAs transcribed from a gene, used to identify where or when a gene is transcribed in an organism • Reverse transcriptase-polymerase chain reaction (RT-PCR) • Reverse transcriptase is added to mRNA to make cDNA, which serves as a template for PCR amplification of the gene of interest

  38. Fig. 20-13 • TECHNIQUE • 1 • cDNA synthesis • mRNAs • cDNAs • Primers • 2 • PCR amplification • -globingene • 3 • Gel electrophoresis • Embryonic stages • RESULTS • 1 2 3 4 5 6

  39. IX. Cloning Animals • Nuclear transplantation = nucleus of an unfertilized egg is replaced w/ nucleus of a differentiated cell • Experiments, w/ frog embryos, have shown a transplanted nucleus can often support normal development of the egg • However, the older the donor nucleus, the lower the percentage of normally developing tadpoles

  40. Fig. 20-17 • Frog egg cell • Frog embryo • Frog tadpole • EXPERIMENT • UV • Fully differ- • entiated • (intestinal) cell • Less differ-entiated cell • Donornucleustrans-planted • Donor • nucleus • trans- • planted • Enucleated • egg cell • Egg with donor nucleus • activated to begin • development • RESULTS • Most develop • into tadpoles • Most stop developing • before tadpole stage

  41. Fig. 20-18 • TECHNIQUE • 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

  42. A. Problems with Animal Cloning • Only a small percentage of cloned embryos have developed normally to birth • Epigenetic changes (acetylation of histones or methylation of DNA) must be reversed in nucleus from a donor animal for genes to be expressed or repressed appropriately for development

  43. X. Medical Applications • Gene therapy = alteration of an afflicted individual’s genes • Vectors are used for delivery of genes into specific types of cells, for example bone marrow, lung tissue (CFTR protein)

  44. Fig. 20-22 • Clonedgene • Insert RNA version of normal alleleinto retrovirus. • 1 • Viral RNA • Let retrovirus infect bone marrow cellsthat have been removed from thepatient and cultured. • 2 • Retroviruscapsid • Viral DNA carrying the normalallele inserts into chromosome. • 3 • Bonemarrowcell frompatient • Bonemarrow • Inject engineeredcells into patient. • 4

  45. XI. Genetic Engineering in Plants • Agricultural scientists have given a number of crop plants genes for desirable traits • Ti plasmid = most commonly used vector for introducing new genes into plant cells • Used to transfer herbicide resistance, inc resistance to pests, inc resistance to salinity, and improved nutritional value

  46. Fig. 20-25 • TECHNIQUE • Agrobacterium tumefaciens • Tiplasmid • Site whererestrictionenzyme cuts • T DNA • RESULTS • DNA withthe geneof interest • RecombinantTi plasmid • Plant with new trait

  47. You should now be able to: • Describe the natural function of restriction enzymes and explain how they are used in recombinant DNA technology • Outline the procedures for cloning a eukaryotic gene in a bacterial plasmid • Define and distinguish between genomic libraries using plasmids, phages, and cDNA • Describe the polymerase chain reaction (PCR) and explain the advantages and limitations of this procedure

  48. Explain how gel electrophoresis is used to analyze nucleic acids and to distinguish between two alleles of a gene • Describe and distinguish between the Southern blotting procedure, Northern blotting procedure, and RT-PCR • Distinguish between gene cloning, cell cloning, and organismal cloning • Describe how nuclear transplantation was used to produce Dolly, the first cloned sheep

  49. Describe the application of DNA technology to the diagnosis of genetic disease, the development of gene therapy, vaccine production, and the development of pharmaceutical products • Define a SNP and explain how it may produce a RFLP • Explain how DNA technology is used in the forensic sciences

  50. Discuss the safety and ethical questions related to recombinant DNA studies and the biotechnology industry

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