1. Biotechnology Chapter 17

2. 2 DNA Manipulation The molecular biology revolution started with the discovery of restriction endonucleases -Enzymes that cleave DNA at specific sites These enzymes are significant in two ways 1. Allow a form of physical mapping that was previously impossible 2. Allow the creation of recombinant DNA molecules (from two different sources)

3. 3 DNA Manipulation Restriction enzymes recognize DNA sequences termed restriction sites There are two types of restriction enzymes: -Type I = Cut near the restriction site -Rarely used in DNA manipulation -Type II = Cut at the restriction site -The sites are palindromes -Both strands have same sequence when read 5 ’ to 3 ’

4. 4 DNA Manipulation Type II enzymes produce staggered cuts that generate “sticky ends” -Overhanging complementary ends Therefore, fragments cut by the same enzyme can be paired DNA ligase can join the two fragments forming a stable DNA molecule

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6. 6 Gel Electrophoresis A technique used to separate DNA fragments by size The gel (agarose or polyacrylamide) is subjected to an electrical field The DNA, which is negatively-charged, migrates towards the positive pole -The larger the DNA fragment, the slower it will move through the gel matrix DNA is visualized using fluorescent dyes

7. 7 Restriction endonuclease 1 cut site Restriction endonuclease 2 cut site Reaction 1 Reaction 2 Reaction 3 Restriction endonuclease 3 Short segmentLong segment Medium segment Mixture of DNA fragments of different sizes in solution placed at the top of “lanes” in the gel Gel Lane Anode + Cathode - Power source Short segmentLong segment Reaction 2 Reaction 1 Reaction 3 Shorter fragments Longer fragments Visualizing Stained Gel Gel is stained with a dye to allow the fragments to be visualized. DNA samples are cut with restriction enzymes in three different reactions producing different patterns of fragments Samples from the restriction enzyme digests are introduced into the gel. Electric current is applied causing fragments to migrate through the gel. Restriction Enzyme Digestion Gel Electrophoresis a.b. c. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Buffer

8. 8 Transformation Transformation is the introduction of DNA from an outside source into a cell Natural transformation occurs in many species -However, not in E. coli, which is used routinely in molecular biology labs -Artificial transformation techniques have been developed to introduce foreign DNA into it

9. 9 Molecular Cloning A clone refers to a genetically identical copy Molecular cloning is the isolation of a specific DNA sequence (usually protein-encoding) -Sometimes called gene cloning The most flexible and common host for cloning is E. coli Propagation of DNA in a host cell requires a vector

10. 10 Vectors Plasmids are small, circular extrachromosomal DNA molecules -Used for cloning small pieces of DNA -Have three important components 1. Origin of replication 2. Selectable marker 3. Multiple cloning site (MCS)

11. 11 Vectors

12. 12 Vectors Phage vectors are modified bacterial viruses -Most based on phage lambda ( ) of E. coli -Used to clone inserts up to 40 Kbp -Have two features not shared with plasmid vectors -They kill their host cells -They have linear genomes -Middle replaced with inserted DNA

13. 13 Vectors

14. 14 Vectors Artificial chromosomes -Used to clone very large DNA fragments -Bacterial artificial chromosomes (BACs) -Yeast artificial chromosomes (YACs)

15. 15 DNA Libraries A collection of DNA fragments from a specific source that has been inserted into host cells A genomic library represents the entire genome A cDNA library represents only the expressed part of the genome -Complementary DNA (cDNA) is synthesized from isolated mRNA using the enzyme reverse transcriptase

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17. 17 5´ cap Eukaryotic DNA template exons introns 12341234 Transcription Primary RNA transcript 5´ cap Mature RNA transcript 3´ poly-A tail Degraded mRNA 3´ poly-A tail mRNA–cDNA hybrid Isolation of mRNA Addition of reverse transcriptase Addition of mRNA- degrading enzymes DNA polymerase Double-stranded cDNA with no introns Reverse transcriptase Reverse transcriptase utilizes mRNA to create cDNA. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Introns are cut out, and coding regions are spliced together.

18. 18 DNA Libraries Molecular hybridization is a technique used to identify specific DNAs in complex mixtures -A known single-stranded DNA or RNA is labeled -It is then used as a probe to identify its complement via specific base-pairing -Also termed annealing

19. 19 DNA Libraries Molecular hybridization is the most common way of identifying a clone in a DNA library -This process involves three steps: 1. Plating the library 2. Replicating the library 3. Screening the library

20. 20 Film Filter paper 1. Colonies of plasmid containing bacteria, each containing a single DNA from the library, are grown on agar. 3. The filter is washed with a solution to break the cells open and denature the DNA, which sticks to the filter at the site of each colony. The filter is incubated with a radioactively labeled probe that can form hybrids with complementary DNA in the gene of interest. 4. The only sites on the filter that will retain probe DNA will contain DNA complementary to the probe. These represent the sites of colonies containing the gene of interest. 5. A comparison with the original plate identifies the colony containing the 2. A replica of the plate is made by pressing a piece of filter paper against the agar and bacterial colonies. Some cells from each colony adhere to the filter.

21. 21 DNA Analysis Restriction maps -Molecular biologists need maps to analyze and compare cloned DNAs -The first maps were restriction maps -Initially, they were created by enzyme digestion & analysis of resulting patterns -Many are now generated by computer searches for cleavage sites

22. 22 DNA Analysis Southern blotting -A sample DNA is digested by restriction enzymes & separated by gel electrophoresis -Gel is transferred (“blotted”) onto a nitrocellulose filter -Then hybridized with a cloned, radioactively-labeled DNA probe -Complementary sequences are revealed by autoradiography

23. 23 3. DNA in the gel is transferred, or “blotted,” onto the nitrocellulose. 4.Nitrocellulose with bound DNA is incubated with radioactively labeled nucleic acids and is then rinsed. 5. Photographic film is laid over the filter and is exposed only in areas that contain radioactivity (autoradiography). Bands on the film represent DNA in the gel that is complementary to the probe sequence. Size markers Hybridized nucleic acids Film Gel Buffer Sponge Nitrocellulose filter Gel 1. Electrophoresis is performed, using radioactively labeled markers as a size guide in the first lane. Test nucleic acids Radioactively labeled markers with specific sizes Electrophoretic gel Electrophoresis DNA fragments within bands Radioactive probe (single- stranded DNA) 2. The gel is covered with a sheet of nitrocellulose and placed in a tray of buffer on top of a sponge. Alkaline chemicals in the buffer denature the DNA into single strands. The buffer wicks its way up through the gel and nitrocellulose into a stack of paper towels placed on top of the nitrocellulose. Stack of paper towels —TTACC— —AATGG— Sealed container Nitrocellulose paper now contains nucleic acid “print” Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

24. 24 DNA Analysis Northern blotting -mRNA is electrophoresed and then blotted onto the filter Western blotting -Proteins are electrophoresed and then blotted onto the filter -Detection requires an antibody that can bind to one protein

25. 25 DNA Analysis RFLP analysis -Restriction fragment length polymorphisms (RFLPs) are generated by point mutations or sequence duplications -These fragments are often not identical in different individuals -Can be detected by Southern blotting

26. 26 a. Three different DNA duplexes b. Cut DNA c. Gel electrophoresis of restriction fragments Original Sequence of Restriction Sites (no mutations) Point Mutations Change the Sequence of Restriction Sites Sequence Repetitions Can Occur Between Restriction Sites Larger fragments Smaller fragments restriction enzyme cutting sites Sequence duplication Single base-pair change + + + + + – – – Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

27. 27 DNA Analysis DNA fingerprinting -An identification technique used to detect differences in the DNA of individuals -Makes use of a variety of molecular procedures, including RFLP analysis -First used in a US criminal trial in 1987 -Tommie Lee Andrews was found guilty of rape

28. 28 DNA Analysis

29. 29 DNA Analysis DNA sequencing -A set of nested fragments is generated -End with known base -Separated by high- resolution gel electrophoresis, resulting in a “ladder” -Sequence is read from the bottom up

30. 30 DNA Analysis DNA sequencing -The enzymatic method was developed by Frederick Sanger -Dideoxynucleotides are used as chain terminators in DNA synthesis reactions

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32. 32 DNA Analysis DNA sequencing -The enzymatic technique is powerful but is labor intensive and time-consuming -The development of automated techniques made sequencing faster and more practical -Fluorescent dyes are used instead of radioactive labels -Reaction is done in one tube -Data are assembled by a computer

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34. 34 DNA Analysis Polymerase chain reaction (PCR) -Developed by Kary Mullis -Allows the amplification of a small DNA fragment using primers that flank the region -Each PCR cycle involves three steps: 1. Denaturation (high temperature) 2. Annealing of primers (low temperature) 3. DNA synthesis (intermediate temperature) -Taq polymerase

35. 35 DNA is denatured into single strands 5´ 3´ 5´3´ 5´ 3´5´ Primers anneal to DNA 5´3´ 5´ Taq DNA polymerase 5´3´ 5´3´ 5´ 3´5´ 3´ 5´3´ 5´3´ 5´3´ 5´3´ 5´3´ 5´3´ 5´3´ 5´ 3´5´ 3´5´ 3´5´ 3´5´ 3´5´ 3´5´ 3´5´ 3´ 5´3´ 5´3´ 5´3´ 5´ 3´5´ 3´5´ 3´5´ PCR machine Cycle 2: 4 copies Cycle 3: 8 copies 1. Sample is first heated to denature DNA. 2. DNA is cooled to a lower temperature to allow annealing of primers. 3. DNA is heated to 72°C, the optimal temperature for Taq DNA polymerase to extend primers. DNA segment to be amplified After 20 cycles, a single fragment produces over one million (2 20 ) copies!

36. 36 DNA Analysis Polymerase chain reaction (PCR) -Has revolutionized science and medicine because it allows the investigation of minute samples of DNA -Forensics -Detection of genetic defects in embryos -Analysis of mitochondrial DNA from early human species

37. 37 DNA Analysis Yeast two-hybrid system -Used to study protein-protein interactions -Gal4 is a transcriptional activator with a modular structure -The Gal4 gene is split into two vectors -Baitvector: has DNA-binding domain -Prey vector: has transcription-activating domain -Neither of these alone can activate transcription

38. 38 DNA Analysis Yeast two-hybrid system -When other genes are inserted into these vectors, they produce fusion proteins -Contain part of Gal4 and the protein of interest -If the proteins being tested interact, Gal4 function will be restored -A reporter gene will be expressed -Detected by an enzyme assay

39. 39 Fusion proteins Bait vector Bait protein Prey vector Prey protein Reporter gene Yeast nucleus Yeast cell Inserted DNA Gal4 protein RNA polymerase DNADNA- binding domain Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Transcription- activating domain DNA- binding domain

40. 40 Genetic Engineering Has generated excitement and controversy Expression vectors contain the sequences necessary to express inserted DNA in a specific cell type Transgenic animals contain genes that have been inserted without the use of conventional breeding

41. 41 Genetic Engineering In vitro mutagenesis -Ability to create mutations at any site in a cloned gene -Has been used to produce knockout mice, in which a known gene is inactivated -The effect of loss of this function is then assessed on the entire organism -An example of reverse genetics

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43. 43 Medical Applications Human proteins -Medically important proteins can be produced in bacteria -Human insulin -Interferon -Atrial peptides -Tissue plasminogen activator -Human growth hormone

44. 44 Medical Applications

45. 45 Medical Applications Vaccines -Subunit vaccines: Genes encoding a part of the protein coat are spliced into a fragment of the vaccinia (cowpox) genome -DNA vaccines: Depend on the cellular immune response (not antibodies)

46. 46 Medical Applications Human immune response Gene specifying herpes simplex surface protein Harmless vaccinia (cowpox) virus Herpes simplex virus 2. Herpes simplex gene is isolated. 3. Vaccinia DNA is extracted and cleaved. 4. Fragment containing surface gene combines with cleaved vaccinia DNA. 5. Harmless engineered virus (the vaccine) with surface like herpes simplex is injected into the human body. 6. Antibodies directed against herpes simplex viral coat are made. 1. DNA is extracted.

47. 47 Medical Applications Gene therapy -Adding a functional copy of a gene to correct a hereditary disorder -Severe combined immunodeficiency disease (SCID) illustrates both the potential and the problems -Successful at first, but then patients developed a rare leukemia

48. 48 Agricultural Applications Ti (tumor-inducing) plasmid is the most used vector for plant genetic engineering -Obtained from Agrobacterium tumefaciens, which normally infects broadleaf plants -However, bacterium does not infect cereals such as corn, rice and wheat

49. 49 Gene of interest Agrobacterium Plasmid Plant nucleus 1. Plasmid is removed and cut open with restriction endonuclease. 2. A gene of interest is isolated from the DNA of another organism and inserted into the plasmid. The plasmid is put back into the Agrobacterium. 3. When used to infect plant cells, Agrobacterium duplicates part of the plasmid and transfers the new gene into a chromosome of the plant cell. 4. The plant cell divides, and each daughter cell receives the new gene. These cultured cells can be used to grow a new plant with the introduced gene. Agricultural Applications

50. 50 Agricultural Applications Gene guns -Uses bombardment with tiny gold particles coated with DNA -Possible for any species -However, the copy number of inserted genes cannot be controlled

51. 51 Agricultural Applications Herbicide resistance -Broadleaf plants have been engineered to be resistant to the herbicide glyphosate -This allows for no-till planting

52. 52 Agricultural Applications Pest resistance -Insecticidal proteins have been transferred into crop plants to make them pest-resistant -Bt toxin from Bacillus thuringiensis Golden rice -Rice that has been genetically modified to produce  -carotene (provitamin A) -Converted in the body to vitamin A

53. 53 Agricultural Applications Daffodil phytoene synthase gene (psy) psycrtIlcy Phytoene synthase Carotene desaturase  -Cyclase Genes introduced into rice genome Expression in endosperm GGPPPhytoeneLycopene  -Carotene (Provitamin A) Bacterial carotene desaturase gene (crtI) Daffodil lycopene  -cyclase gene (lcy) Rice chromosome

54. 54 Agricultural Applications Adoption of genetically modified (GM) crops has been resisted in some areas because of questions about: -Crop safety for human consumption -Movement of genes into wild relatives -Loss of biodiversity

55. 55 Agricultural Applications Biopharming -Transgenic plants are used to produce pharmaceuticals -Human serum albumin -Recombinant subunit vaccines -Against Norwalk and rabies viruses -Recombinant monoclonal antibodies -Against tooth decay-causing bacteria

56. 56 Agricultural Applications Transgenic animal technology has not been as successful as that in plants -One interesting example is the EnviroPig -Engineered to carry the gene for the enzyme phytase -Breaks down phosphorus in feed -Reduces excretion of harmful phosphates in the environment

57. 57 Agricultural Applications