Recombinant DNA Technology 1

The Role of Recombinant DNA technology in Biotechnology Recombinant 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 2

Overview of recombinant DNA technology 3 Figure 8.1

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 4

The Tools of Recombinant DNA Technology The Use of Reverse Transcriptase to Synthesize cDNA ◦Isolated from retroviruses ◦Uses RNA template to transcribe molecule of cDNA ◦Easier to isolate mRNA molecule for desired protein first ◦mRNA of eukaryotes has introns removed  Allows cloning in prokaryotic cells 5

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 6

The Tools of Recombinant DNA Technology Restriction Enzymes ◦Bacterial enzymes that cut DNA molecules only at restriction sites ◦Categorized into two groups based on type of cut  Cuts with sticky ends  Cuts with blunt ends 7

Actions of restriction enzymes 8 Figure 8.2

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 9

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

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 use to amplify genome of unknown pathogen  Amplified DNA from Bacillus anthracis spores in 2001 to identify source of spores 11

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 12

Polymerase chain reaction (PCR) 13 Figure 8.5a

Polymerase chain reaction (PCR) 14 Figure 8.5b

Techniques of Recombinant DNA Technology 15

Techniques of Recombinant DNA Technology Selecting a Clone of Recombinant Cells ◦Must find clone containing DNA of interest ◦Probes are used 16

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 17

Gel electrophoresis 18 Figure 8.6

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 – used to detect RNA ◦Uses of Southern blots  Genetic “fingerprinting”  Diagnosis of infectious disease  Demonstrate incidence and prevalence of organisms that cannot be cultured 19

The Southern blot technique 20 Figure 8.7

Techniques of Recombinant DNA Technology DNA Microarrays ◦Consist of molecules of immobilized single- stranded DNA ◦Fluorescently labeled DNA washed over array will adhere only at locations where there are complementary DNA sequences ◦Variety of scientific uses of DNA microarrays  Monitoring gene expression  Diagnosis of infection  Identification of organisms in an environmental sample 21

DNA microarray 22 Figure 8.8

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 23

Artificial methods of inserting DNA into cells 24 Figure 8.9a/b

Artificial methods of inserting DNA into cells 25 Figure 8.9c/d

Applications of Recombinant DNA Technology Applications of Recombinant DNA Technology Genetic Mapping ◦Locating genes on a nucleic acid molecule ◦Provides useful facts concerning metabolism, growth characteristics, and relatedness to others Locating Genes ◦Until 1970, genes identified by labor-intensive methods ◦Simpler and universal methods now available ◦Restriction fragmentation ◦Fluorescent in situ hybridization (FISH) 26

Fluorescent in situ hybridization 27 Figure 8.10

Automated DNA sequencing 28 Figure 8.11

Applications of Recombinant DNA Technology Applications of Recombinant DNA Technology Environmental 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  Determined that methane-producing archaea are a problem in rice agriculture 29

Applications of Recombinant DNA Technology Applications of Recombinant DNA Technology Pharmaceutical and Therapeutic Applications ◦Protein synthesis  Creation of synthetic peptides for cloning ◦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 30

Applications of Recombinant DNA Technology 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 pathogen’s DNA in blood or tissues ◦DNA fingerprinting  Identifying individuals or organisms by their unique DNA sequence 31

DNA fingerprinting 32 Figure 8.12

Applications of Recombinant DNA Technology Applications of Recombinant DNA Technology Pharmaceutical and Therapeutic Applications ◦Gene therapy  Missing or defective genes replaced with normal copies  Some patients’ immune systems react negatively ◦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 33

Applications of Recombinant DNA Technology Applications of Recombinant DNA Technology Agricultural Applications ◦Production of transgenic organisms  Recombinant plants and animals altered by addition of genes from other organisms 34

Applications of Recombinant DNA Technology Applications of Recombinant DNA Technology Agricultural Applications ◦Herbicide resistance  Gene from Salmonella conveys resistance to glyphosate (Roundup) ◦ Farmers can kill weeds without killing crops ◦Salt tolerance  Scientists have removed gene for salt tolerance and inserted into tomato and canola plants  Transgenic plants survive, produce fruit, and remove salt from soil 35

Applications of Recombinant DNA Technology Applications of Recombinant DNA Technology Agricultural Applications ◦Freeze resistance  Crops sprayed with genetically modified bacteria can tolerate mild freezes ◦Pest resistance  Bt toxin ◦ Naturally occurring toxin only harmful to insects ◦ Organic farmers used to reduce insect damage to crops  Gene for Bt toxin inserted into various crop plants  Genes for Phytophthora resistance inserted into potato crops 36

Applications of Recombinant DNA Technology Applications of Recombinant DNA Technology Agricultural Applications ◦Improvements in nutritional value and yield  Tomatoes allowed to ripen on vine and shelf life increased ◦ Gene for enzyme that breaks down pectin suppressed  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 37

The Ethics and Safety of Recombinant DNA Technology Supremacist view – humans are of greater value than animals 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 38

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 39

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”? 40