Chapter 8 Recombinant DNA Technology. 10/1/ MDufilho

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1 Chapter 8 Recombinant DNA Technology 10/1/ MDufilho

2 The Role of Recombinant DNA Technology in Biotechnology Biotechnology? Recombinant deoxyribonucleic acid (DNA) technology Intentionally modifying genomes of organisms for practical purposes Three goals: Eliminate undesirable phenotypic traits Combine beneficial traits of two or more organisms Create organisms that synthesize products humans need 10/1/2017 MDufilho 2

3 Figure 8.1 Overview of recombinant DNA technology. Bacterial cell DNA containing gene of interest Bacterial chromosome Plasmid Isolate plasmid. Gene of interest Enzymatically cleave DNA into fragments. Isolate fragment with the gene of interest. Insert gene into plasmid. Insert plasmid and gene into bacterium. Culture bacteria. Harvest copies of gene to insert into plants or animals. Harvest proteins coded by gene. Eliminate undesirable phenotypic traits. Create beneficial combination of traits. Produce vaccines, antibiotics, hormones, or enzymes. 10/1/2017 MDufilho 3

4 The Tools of Recombinant DNA Technology Mutagens Physical and chemical agents that produce mutations Scientists utilize mutagens to: create changes in microbes' genomes to change phenotypes. select for and culture cells with beneficial characteristics. Mutated genes alone can be isolated 10/1/2017 MDufilho 4

5 The Tools of Recombinant DNA Technology The Use of Reverse Transcriptase to Synthesize complementary DNA (cdna) Isolated from retroviruses Uses ribonucleic acid (RNA) template to transcribe molecule of cdna Easier to isolate mitochondrial RNA (mrna) molecule for desired protein first cdna generated from mrna of eukaryotes has introns removed Allows cloning in prokaryotic cells 10/1/2017 MDufilho 5

6 The Tools of Recombinant DNA Technology Synthetic Nucleic Acids Molecules of DNA and RNA produced in cell-free solutions Uses of synthetic nucleic acids: Elucidating the genetic code Creating genes for specific proteins Synthesizing DNA and RNA probes to locate specific sequences of nucleotides Synthesizing antisense nucleic acid molecules Synthesizing polymerase chain reaction (PCR) primers 10/1/2017 MDufilho 6

7 The Tools of Recombinant DNA Technology Restriction Enzymes Bacterial enzymes that cut DNA molecules only at restriction sites Restriction site usually sequences palindromes Categorized into two groups based on type of cut: Cuts with sticky ends Cuts with blunt ends 10/1/2017 MDufilho 7

8 Figure 8.2 Actions of representative restriction enzymes. Restriction site (palindrome) Restriction enzyme Restriction enzyme 1 Restriction enzyme Production of sticky ends Sticky ends Blunt ends Production of blunt ends Ligase Restriction fragments from two different organisms cut by the same restriction enzyme Ligase Recombinant DNA molecules Recombinants using blunt ends Recombinant DNA molecules Recombinants using sticky ends 10/1/2017 MDufilho 8

9 Recombinant DNA Technology PLAY Recombinant DNA Technology 10/1/2017 MDufilho 9

10 The Tools of Recombinant DNA Technology Vectors Nucleic acid molecules that deliver a gene into a cell Useful properties: Small enough to manipulate in a lab Survive inside cells Contain recognizable genetic marker Ensure genetic expression of gene Include viral genomes, transposons, and plasmids 10/1/2017 MDufilho 10

11 Figure 8.3 An example of the process for producing a recombinant vector. Antibioticresistance gene Restriction site mrna for human growth hormone (HGH) Reverse transcription Plasmid (vector) Restriction enzyme Restriction enzyme cdna for HGH Sticky ends Gene for human growth hormone Ligase Recombinant plasmid Introduce recombinant plasmid into bacteria. Bacterial chromosome Recombinant plasmid Inoculate bacteria on media containing antibiotic. Bacteria containing the plasmid with HGH gene survive because they also have resistance gene. 10/1/2017 MDufilho 11

12 The Tools of Recombinant DNA Technology Gene Libraries A collection of bacterial or phage clones Each clone in library often contains one gene of an organism's genome Library may contain all genes of a single chromosome Library may contain set of cdna complementary to mrna 10/1/2017 MDufilho 12

13 Figure 8.4 Production of a gene library. Genome Isolate genome of organism Generate fragments using restriction enzymes. Insert each fragment into a vector Introduce vectors into cells Culture recombinant cells; descendants are clones. 10/1/2017 MDufilho

14 Techniques of Recombinant DNA Technology Multiplying DNA in vitro: The Polymerase Chain Reaction (PCR) Large number of identical molecules of DNA produced in vitro Critical to amplify DNA in variety of situations Epidemiologists used PCR to determine that two separate Ebola outbreaks occurred in Africa in 2014 Amplified DNA from Bacillus anthracis spores in 2001 to identify source of spores 10/1/2017 MDufilho 14

15 Techniques of Recombinant DNA Technology Multiplying DNA in vitro: The Polymerase Chain Reaction (PCR) Repetitive process consisting of three steps: Denaturation Priming Extension Can be automated using a thermocycler 10/1/2017 MDufilho 15

16 Techniques of Recombinant DNA Technology Selecting a Clone of Recombinant Cells Must find clone containing DNA of interest Probes are used 10/1/2017 MDufilho 16

17 Techniques of Recombinant DNA Technology Separating DNA Molecules: Gel Electrophoresis and the Southern Blot Gel electrophoresis: Separates molecules based on electrical charge, size, and shape Allows scientists to isolate DNA of interest Negatively charged DNA drawn toward positive electrode Agarose makes up gel; acts as molecular sieve Smaller fragments migrate faster and farther than larger ones Determine size by comparing distance migrated to standards 10/1/2017 MDufilho 17

18 Figure 8.6 Gel electrophoresis. Wells ( ) A B C D E (50) (40) (35) Electrophoresis chamber filled with buffer solution Agarose gel (+) (15) (10) (5) DNA a Movement of DNA b Wire Lane of DNA fragments of known sizes (kilobase pairs) 10/1/2017 MDufilho 18

19 Techniques of Recombinant DNA Technology Separating DNA Molecules: Gel Electrophoresis and the Southern Blot Southern blot DNA transferred from gel to nitrocellulose membrane Probes used to localize DNA sequence of interest Northern blot similar technique used to detect RNA Uses of Southern blot Genetic fingerprinting Diagnosing infectious disease Demonstrating the presence of organisms that cannot be cultured 10/1/2017 MDufilho 19

20 Techniques of Recombinant DNA Technology Inserting DNA into Cells Goal of DNA technology is insertion of DNA into cell Natural methods: Transformation Transduction Conjugation Artificial methods: Electroporation Protoplast fusion Injection gene gun and microinjection 10/1/2017 MDufilho 20

21 Figure 8.8a-b Artificial methods of inserting DNA into cells. Pores in wall and membrane Electroporation Chromosome Electrical field applied Competent cell DNA from another source Cell synthesizes new wall Recombinant cell Cell walls Cell synthesizes new wall Enzymes remove cell walls Polyethylene glycol Recombinant cell New cell Protoplasts Fused protoplasts Protoplast fusion 10/1/2017 MDufilho 21

22 Figure 8.8c-d Artificial methods of inserting DNA into cells. Micropipette containing DNA Target cell's nucleus Target cell Suction tube to hold target cell in place Blank.22- caliber shell Nylon projectile Vent Plate to stop nylon projectile DNA-coated beads Target cell Gene gun Nylon projectile Microinjection 10/1/2017 MDufilho 22

23 Applications of Recombinant DNA Technology Genetic Mapping Locating genes on a nucleic acid molecule Until 1970, genes identified by labor-intensive methods Simpler and universal methods now available Restriction fragmentation Provides useful facts concerning metabolism, growth characteristics, and relatedness to others 10/1/2017 MDufilho 23

24 Applications of Recombinant DNA Technology Microbial Communities Studies Most microorganisms have never been grown in a laboratory Scientists know them only by their DNA fingerprints Allowed identification of over 500 species of bacteria from human mouths Next-generation sequencing allows for the determination of all of the members of a microbiome 10/1/2017 MDufilho 24

25 Applications of Recombinant DNA Technology Pharmaceutical and Therapeutic Applications Protein synthesis Creation of synthetic proteins by bacteria and yeast cells Vaccines Production of safer vaccines Subunit vaccines Introduce genes of pathogens into common fruits and vegetables Injecting humans with plasmid-carrying gene from pathogen Humans synthesize pathogen's proteins 10/1/2017 MDufilho 25

26 Applications of Recombinant DNA Technology Pharmaceutical and Therapeutic Applications Genetic screening DNA microarrays used to screen individuals for inherited disease caused by mutations Can also identify viral DNA in blood or tissues Gene therapy Missing or defective genes replaced with normal copies Difficult to get a functioning gene into enough cells to affect the disease 10/1/2017 MDufilho 26

27 Applications of Recombinant DNA Technology Pharmaceutical and Therapeutic Applications Medical diagnosis Patient specimens can be examined for presence of gene sequences unique to certain pathogens Xenotransplants Animal cells, tissues, or organs introduced into human body Biomedical Animal Models Animals are used in biomedical research to study diseases and develop new diagnostic and therapeutic procedures 10/1/2017 MDufilho 27

28 Applications of Recombinant DNA Technology Agricultural Applications Production of transgenic organisms Recombinant plants and animals altered by addition of genes from other organisms Also called genetically modified organisms (GMOs) Herbicide tolerance Gene from Agrobacterium tumefaciens conveys resistance to glyphosate (Roundup) Farmers can kill weeds without killing crops Salt tolerance Scientists have inserted a gene for salt tolerance into tomato and canola plants Transgenic plants survive, produce fruit, and remove salt from soil 10/1/2017 MDufilho 28

29 Applications of Recombinant DNA Technology Agricultural Applications Freeze resistance Crops sprayed with genetically modified bacteria can tolerate mild freezes Pest resistance Bacillus thuringiensis (Bt) toxin Naturally occurring toxin only harmful to insects Organic farmers use to reduce insect damage to crops Gene for Bt toxin inserted into various crop plants Genes for Phytophthora resistance inserted into potato crops 10/1/2017 MDufilho 29

30 Applications of Recombinant DNA Technology Agricultural Applications Improvements in nutritional value and yield Enzyme that breaks down pectin suppressed in some tomatoes Allows tomatoes to ripen on vine and increases shelf life Bovine growth hormone (BGH) allows cattle to gain weight more rapidly Have meat with lower fat content and produce 10% more milk Gene for β-carotene (vitamin A precursor) inserted into rice Scientists considering transplanting genes coding for entire metabolic pathways 10/1/2017 MDufilho 30

31 The Ethics and Safety of Recombinant DNA Technology Long-term effects of transgenic manipulations are unknown Unforeseen problems arise from every new technology and procedure Natural genetic transfer could deliver genes from transgenic plants and animals into other organisms Transgenic organisms could trigger allergies or cause harmless organisms to become pathogenic 10/1/2017 MDufilho 31

32 The Ethics and Safety of Recombinant DNA Technology Studies have not shown any risks to human health or environment Standards imposed on labs involved in recombinant DNA technology Can create biological weapons using same technology 10/1/2017 MDufilho 32

33 The Ethics and Safety of Recombinant DNA Technology Ethical issues Routine screenings? Who should pay? Genetic privacy rights? Profits from genetically altered organisms? Required genetic screening? Forced correction of "genetic abnormalities"? 10/1/2017 MDufilho 33