1. Chapter 20 Biotechnology

2. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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 DNA of chromosome Cell containing gene of interest Gene inserted into plasmid Plasmid put into bacterial cell Recombinant DNA (plasmid) Recombinant bacterium Bacterial chromosome Bacterium Gene of interest Plasmid 2 1 2

8. Fig. 20-2b Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Gene of Interest Protein expressed by gene of interest Basic research and various applications Copies of gene Protein harvested Basic research on gene Basic research on protein 4 Recombinant bacterium Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hor- mone treats stunted growth 3

9. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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 DNA Sticky end Restriction enzyme cuts sugar-phosphate backbones. 5353 3535 1 One possible combination Recombinant DNA molecule DNA ligase seals strands. 3 DNA fragment added from another molecule cut by same enzyme. Base pairing occurs. 2

11. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings – 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 Bacterial cell Bacterial plasmid lacZ gene Hummingbird cell Gene of interest Hummingbird DNA fragments Restriction site Sticky ends amp R gene TECHNIQUE Recombinant plasmids Nonrecombinant plasmid Bacteria carrying plasmids RESULTS Colony carrying non- recombinant plasmid with intact lacZ gene One of many bacterial clones Colony carrying recombinant plasmid with disrupted lacZ gene

15. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings C 3 5 CCGGGAAATTT 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 G 5 3 …… GGCCCTTTAAA

16. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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 Probe DNA Radioactively labeled probe molecules Film Nylon membrane Multiwell plates holding library clones Location of DNA with the complementary sequence Gene of interest Single-stranded DNA from cell Nylon membrane TECHNIQUE

18. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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 5 Genomic DNA TECHNIQUE Cycle 1 yields 2 molecules Denaturation Annealing Extension Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence Target sequence Primers New nucleo- tides 3 3 3 3 5 5 5 1 2 3

24. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings DNA Cloning - Andersen

25. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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 Mixture of DNA mol- ecules of different sizes Power source Longer molecules Shorter molecules Gel Anode Cathode TECHNIQUE RESULTS 1 2 + + – –

28. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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 allele Sickle-cell allele Large fragment (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles 201 bp 175 bp 376 bp (a) Dde I restriction sites in normal and sickle-cell alleles of  -globin gene Normal  -globin allele Sickle-cell mutant  -globin allele Dde I Large fragment 376 bp 201 bp 175 bp Dde I

30. Gel Electrophoresis Virtual Lab

31. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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 Nitrocellulose membrane (blot) Restriction fragments Alkaline solution DNA transfer (blotting) Sponge Gel Heavy weight Paper towels Preparation of restriction fragmentsGel electrophoresis I II III DNA + restriction enzyme III Heterozygote II Sickle-cell allele I Normal  -globin allele 1 32

33. Fig. 20-11b I II III Film over blot Probe detectionHybridization with radioactive probe Fragment from sickle-cell  -globin allele Fragment from normal  -globin allele Probe base-pairs with fragments Nitrocellulose blot 4 5 Radioactively labeled probe for  -globin gene

34. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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 DNA (template strand) TECHNIQUE DNA polymerase Primer DeoxyribonucleotidesDideoxyribonucleotides (fluorescently tagged) dATP dCTP dTTP dGTP ddATP ddCTP ddTTP ddGTP

36. Fig. 20-12b TECHNIQUE RESULTS DNA (template strand) Shortest Labeled strands Longest Shortest labeled strand Longest labeled strand Laser Direction of movement of strands Detector Last base of longest labeled strand Last base of shortest labeled strand

37. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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 RESULTS Gel electrophoresis cDNAs  -globin gene PCR amplification Embryonic stages Primers 1 2 3 4 5 6 mRNAs cDNA synthesis 1 2 3

39. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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 EXPERIMENT Less differ- entiated cell RESULTS Frog embryo Frog egg cell UV Donor nucleus trans- planted Frog tadpole Enucleated egg cell Egg with donor nucleus activated to begin development Fully differ- entiated (intestinal) cell Donor nucleus trans- planted Most develop into tadpoles Most stop developing before tadpole stage

41. Fig. 20-18 TECHNIQUE Mammary cell donor RESULTS Surrogate mother Nucleus from mammary cell Cultured mammary cells Implanted in uterus of a third sheep Early embryo Nucleus removed Egg cell donor Embryonic development Lamb (“Dolly”) genetically identical to mammary cell donor Egg cell from ovary Cells fused Grown in culture 1 3 3 4 5 6 2

42. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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 Bone marrow Cloned gene Bone marrow cell from patient Insert RNA version of normal allele into retrovirus. Retrovirus capsid Viral RNA Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Viral DNA carrying the normal allele inserts into chromosome. Inject engineered cells into patient. 1 2 3 4

45. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 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 Site where restriction enzyme cuts T DNA Plant with new trait Ti plasmid Agrobacterium tumefaciens DNA with the gene of interest Recombinant Ti plasmid TECHNIQUE RESULTS

47. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings You should now be able to: 1.Describe the natural function of restriction enzymes and explain how they are used in recombinant DNA technology 2.Outline the procedures for cloning a eukaryotic gene in a bacterial plasmid 3.Define and distinguish between genomic libraries using plasmids, phages, and cDNA 4.Describe the polymerase chain reaction (PCR) and explain the advantages and limitations of this procedure

48. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 5.Explain how gel electrophoresis is used to analyze nucleic acids and to distinguish between two alleles of a gene 6.Describe and distinguish between the Southern blotting procedure, Northern blotting procedure, and RT-PCR 7.Distinguish between gene cloning, cell cloning, and organismal cloning 8.Describe how nuclear transplantation was used to produce Dolly, the first cloned sheep

49. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 9.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 10.Define a SNP and explain how it may produce a RFLP 11.Explain how DNA technology is used in the forensic sciences

50. Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 12.Discuss the safety and ethical questions related to recombinant DNA studies and the biotechnology industry