Recombinant DNA Technology Chapter 8 Recombinant DNA Technology 10/1/11 MDufilho

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 10/1/11 MDufilho 2

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 10/1/11 MDufilho

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 10/1/11 MDufilho 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 10/1/11 MDufilho 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 10/1/11 MDufilho 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 10/1/11 MDufilho 7

Figure 8.2 Actions of restriction enzymes-overview 10/1/11 MDufilho

The Tools of Recombinant DNA Technology ANIMATION Recombinant DNA Technology 10/1/11 MDufilho 9

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 10/1/11 MDufilho 10

Figure 8.3 Producing a recombinant vector mRNA for human growth hormone (HGH) Antibiotic resistance gene Restriction site Reverse transcription cDNA for HGH Plasmid (vector) Restriction enzyme 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. 10/1/11 MDufilho

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 10/1/11 MDufilho 12

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. 10/1/11 MDufilho

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 10/1/11 MDufilho 14

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 10/1/11 MDufilho 15

Techniques of Recombinant DNA Technology ANIMATION Polymerase Chain Reaction: Components 10/1/11 MDufilho 16

Techniques of Recombinant DNA Technology ANIMATION PCR: The Process 10/1/11 MDufilho . 17

Techniques of Recombinant DNA Technology Selecting a Clone of Recombinant Cells Must find clone containing DNA of interest Probes are used 10/1/11 MDufilho 18

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 10/1/11 MDufilho 19

Figure 8.6 Gel electrophoresis-overview 10/1/11 MDufilho

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 10/1/11 MDufilho 21

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 NaOH. DNA DNA bands Gel The DNA fragments are invisible to the investigators at this stage. 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. Develop film. Developed film 10/1/11 MDufilho

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 of gene expression Diagnosis of infection Identification of organisms in an environmental sample 10/1/11 MDufilho 23

Figure 8.8 DNA microarray-overview 10/1/11 MDufilho

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 10/1/11 MDufilho 25

Figure 8.9a Artificial methods of inserting DNA into cells: electroporation Pores in wall and membrane Chromosome Cell synthesizes new wall Electrical field applied Competent cell Recombinant cell DNA from another source Electroporation 10/1/11 MDufilho

Figure 8.9b Artificial methods of inserting DNA into cells: protoplast fusion Cell walls Cell synthesizes new wall Enzymes remove cell walls Polyethylene glycol Recombinant cell New wall Protoplasts Fused protoplasts Protoplast fusion 10/1/11 MDufilho

Figure 8.9c Artificial methods of inserting DNA into cells: gene gun Blank .22 caliber shell Nylon projectile Vent Plate to stop nylon projectile DNA-coated beads Target cell Protoplasts Nylon projectile Gene gun 10/1/11 MDufilho

Figure 8.9d 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 10/1/11 MDufilho

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) 10/1/11 MDufilho 30

Figure 8.10 FISH 10/1/11 MDufilho

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 10/1/11 MDufilho 32

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 10/1/11 MDufilho 33

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 10/1/11 MDufilho 34

Figure 8.12 DNA fingerprinting 10/1/11 MDufilho

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 10/1/11 MDufilho 36

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 10/1/11 MDufilho 37

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 10/1/11 MDufilho © 2012 Pearson Education Inc. 38

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”? 10/1/11 MDufilho © 2012 Pearson Education Inc. 39