Chapter 8 Recombinant DNA Technology 2012 Pearson Education

The Role of Recombinant DNA Technology in Biotechnology • Recombinant DNA Technology – Intentional modification of organisms’ genomes for practical purposes – Three goals • Eliminate undesirable phenotypic traits • Combine beneficial traits of two or more organisms • Create organisms that synthesize products humans need © 2012 Pearson Education Inc.

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 Eliminate undesirable phenotypic traits Create beneficial combination of traits Harvest proteins coded by gene Produce vaccines, antibiotics, hormones, or enzymes

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 © 2012 Pearson Education Inc.

The Tools of Recombinant DNA Technology • The Use of Reverse Transcriptase to Synthesize c. DNA – Isolated from retroviruses – Uses RNA template to transcribe molecule of c. DNA – Easier to isolate m. RNA molecule for desired protein first – m. RNA of eukaryotes has introns removed • Allows cloning in prokaryotic cells © 2012 Pearson Education Inc.

The Tools of Recombinant DNA Technology • Synthetic Nucleic Acids – Molecules of DNA and RNA produced in cellfree 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 © 2012 Pearson Education Inc.

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 © 2012 Pearson Education Inc.

Figure 8. 2 Actions of restriction enzymes-overview

The Tools of Recombinant DNA Technology ANIMATION Recombinant DNA Technology © 2012 Pearson Education

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 © 2012 Pearson Education Inc.

Figure 8. 3 Producing a recombinant vector m. RNA for human growth hormone (HGH) Antibiotic resistance gene Restriction site Reverse transcription c. DNA for HGH Plasmid (vector) Restriction enzyme 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.

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 c. DNA complementary to m. RNA © 2012 Pearson Education Inc.

Figure 8. 4 Production of a gene library-overview Genome Isolate genome or organism. Generate fragments using restriction enzymes. Insert each fragment into a vector. Introduce vectors into cells. Culture recombinant cells; descendants are clones.

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 © 2012 Pearson Education Inc.

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 © 2012 Pearson Education Inc.

Techniques of Recombinant DNA Technology ANIMATION Polymerase Chain Reaction: Overview © 2012 Pearson Education

Techniques of Recombinant DNA Technology ANIMATION Polymerase Chain Reaction: Components © 2012 Pearson Education

Figure 8. 5 a The use of PCR to replicate DNA, steps 1 -3 Original DNA 3´ molecule 5´ 3´ 5´ Heat to 94°C Denaturation DNA primer Priming Deoxyribonucleotide triphosphates DNA polymerase Cool to 65°C DNA polymerase 5´ 3´ 5´ Extension DNA primer 5´ 3´ 5´ 72°C

Figure 8. 5 b The use of PCR to replicate DNA, step 4 First cycle Second cycle Third cycle Fourth cycle 2 DNA molecules 4 DNA molecules 8 DNA molecules Repeat 16 DNA molecules

Techniques of Recombinant DNA Technology ANIMATION PCR: The Process © 2012 Pearson Education Inc.

Techniques of Recombinant DNA Technology • Selecting a Clone of Recombinant Cells – Must

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 than larger ones – Determine size by comparing distance migrated to standards © 2012 Pearson Education Inc.

Figure 8. 6 Gel electrophoresis-overview

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 © 2012 Pearson Education Inc.

Figure 8. 7 The Southern blot technique-overview DNA molecules Restriction enzymes Restriction fragments Use gel electrophoresis to separate fragments by size; denature DNA into single strands with Na. OH. DNA bands The DNA fragments are invisible to the investigators at this stage. Gel Nitrocellulose membrane Absorbent material Side view Electrophoresis gel Nitrocellulose membrane Absorbent material Nitrocellulose membrane with DNA fragments at same locations as in gel (still invisible) is baked to permanently affix DNA. Add radioactive probes complementary to DNA nucleotide sequence of interest. Probes bind to DNA of interest. Incubate with film; radiation exposes film. Developed film

Techniques of Recombinant DNA Technology • DNA Microarrays – Consist of molecules of immobilized singlestranded 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 of gene expression • Diagnosis of infection • Identification of organisms in an environmental sample © 2012 Pearson Education Inc.

Figure 8. 8 DNA microarray-overview

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 © 2012 Pearson Education Inc.

Figure 8. 9 a Artificial methods of inserting DNA into cells: electroporation Pores in wall and membrane Chromosome Cell synthesizes new wall Electrical field applied Electroporation Recombinant cell Competent cell DNA from another source

Figure 8. 9 b Artificial methods of inserting DNA into cells: protoplast fusion Cell synthesizes new wall Cell walls Enzymes remove cell walls Polyethylene glycol Recombinant cell New wall Protoplasts Protoplast fusion Fused protoplasts

Figure 8. 9 c Artificial methods of inserting DNA into cells: gene gun Blank. 22 Nylon caliber shell projectile Vent Plate to stop nylon projectile Target cell DNA-coated beads Protoplasts Gene gun Nylon projectile

Figure 8. 9 d Artificial methods of inserting DNA into cells: microinjection Micropipette containing DNA Target cell’s nucleus Target cell Suction tube to hold target cell in place Microinjection

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) © 2012 Pearson Education Inc.

Figure 8. 10 FISH

Figure 8. 11 Automated DNA sequencing Nucleotide bases: T G C Signal intensity A 0 100 Number of bases 200

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 © 2012 Pearson Education Inc.

Applications of Recombinant DNA Technology • Pharmaceutical and Therapeutic Applications – Protein synthesis • Creation of synthetic peptides for cloning – Vaccines • Production of safer vaccines • Subunit vaccines • Genes of pathogens introduced into common fruits and vegetables • Injecting humans with plasmid carrying gene from pathogen – Humans synthesize pathogen’s proteins © 2012 Pearson Education Inc.

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 © 2012 Pearson Education Inc.

Figure 8. 12 DNA fingerprinting

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 © 2012 Pearson Education Inc.

Applications of Recombinant DNA Technology • Agricultural Applications – Production of transgenic organisms • Recombinant plants and animals altered by addition of genes from other organisms © 2012 Pearson Education Inc.

Applications of Recombinant DNA Technology • Agricultural Applications – Herbicide tolerance • Gene from Salmonella conveys resistance to glyphosate (Roundup™) – Farmers can kill weeds without killing crops – Salt tolerance • Scientists have inserted gene for salt tolerance into tomato and canola plants • Transgenic plants survive, produce fruit, and remove salt from soil © 2012 Pearson Education Inc.

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 harmful only 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 © 2012 Pearson Education Inc.

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 – Produce 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 © 2012 Pearson Education Inc.

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 © 2012 Pearson Education Inc.

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 © 2012 Pearson Education Inc.

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”? © 2012 Pearson Education Inc.