Recombinant DNA Technology 8 Recombinant DNA Technology

The Role of Recombinant DNA Technology in Biotechnology Biotechnology – the use of microorganisms to make practical products 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

Figure 8.1 Overview of recombinant DNA technology. Bacterial cell DNA containing gene of interest Bacterial chromosome Plasmid 1 Isolate plasmid. Gene of interest 2 Enzymatically cleave DNA into fragments. 3 Isolate fragment with the gene of interest. 4 Insert gene into plasmid. 5 Insert plasmid and gene into bacterium. 6 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

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

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 cDNA generated from mRNA of eukaryotes has introns removed Allows cloning in prokaryotic cells

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

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

Figure 8.2 Actions of restriction enzymes. Restriction site (palindrome) 5 G A A T T C 3 5 C C C G G G 3 5 G T T A A C 3 C T T A A G G G G C C C C A A T T G Restriction enzyme Restriction enzyme 1 Restriction enzyme 2 A A T T 5 C C C G G G 3 5 G T T A A C 3 G C G G G C C C C A A T T G C T T A A G Blunt ends Sticky ends Production of blunt ends Production of sticky ends Ligase A A G G C C T T A T A T 5 C C C A A C 3 5 G T T G G G 3 A A T T C G A T T C G A G G G T T G C A A C C C Recombinant DNA molecules Restriction fragments from two different organisms cut by the same restriction enzyme Recombinants using blunt ends Ligase 5 A A G C T T 3 5 A A G C T T 3 + T T C G A A T T C G A A Recombinant DNA molecules Recombinants using sticky ends

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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

Figure 8.3 An example of the process for producing a recombinant vector. mRNA for human growth hormone (HGH) A A Antibiotic resistance gene T T G C C Restriction site G A T A T Reverse transcription cDNA for HGH Plasmid (vector) 1 Restriction enzyme Restriction enzyme A Sticky ends T T C G A A G C T T HGH A A HGH T T C G A A G Gene for human growth hormone A T C T A A G T T C C G A T A T H H G G 2 H H C T T A A G G A A C T T Ligase A A T T G C C G A T A T H H G G H H T A C T A G G A A C T T Recombinant plasmid 3 Introduce recombinant plasmid into bacteria. Bacterial chromosome Recombinant plasmid 4 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 cDNA complementary to mRNA

Figure 8.4 Production of a gene library. Genome 1 Isolate genome of organism. 2 Generate fragments using restriction enzymes. 1 2 3 4 5 6 7 8 9 10 11 3 Insert each fragment into a vector. 1 2 3 4 5 6 7 8 9 10 11 4 Introduce vectors into cells. 1 2 3 4 5 6 7 8 9 10 11 5 Culture recombinant cells; descendants are clones. 1 2 3 4 5 6 7 8 9 10 11

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

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

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Original DNA molecule 3 3 5 5 Heat to 94C 1 Denaturation Figure 8.5a The use of the polymerase chain reaction (PCR) to replicate DNA. Original DNA molecule 3 3 5 5 Heat to 94C 1 Denaturation DNA primer Deoxyribonucleotide triphosphates 2 Priming DNA polymerase 4 Repeat Cool to 65C DNA polymerase 3 5 5 3 Extension DNA primer 5 5 3 72C

Figure 8.5b The use of the polymerase chain reaction (PCR) to replicate DNA.

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

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

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

Techniques of Recombinant DNA Technology parating 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 blots Genetic "fingerprSeinting" Diagnosis of infectious disease Demonstrate presence of organisms that cannot be cultured

Figure 8.7 The Southern blot technique. DNA molecules Restriction enzymes Restriction fragments Use gel electrophoresis to separate fragments by size; denature DNA into single strands with NaOH. 1 DNA DNA bands Gel The DNA fragments are invisible to the investigators at this stage. Nitrocellulose membrane Absorbent material 2 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. 3 Add radioactive probes complementary to DNA nucleotide sequence of interest. Probes bind to DNA of interest. 4 Incubate with film; radiation exposes film. Develop film. Developed film 5

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

Figure 8.8 DNA microarray.

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

Figure 8.9a-b Artificial methods of inserting DNA into cells. Pores in wall and membrane Chromosome Cell synthesizes new wall Electrical field applied Competent cell Recombinant cell DNA from another source Electroporation Cell walls Cell synthesizes new wall Enzymes remove cell walls Polyethylene glycol Recombinant cell New wall Protoplasts Fused protoplasts Protoplast fusion

Figure 8.9c-d Artificial methods of inserting DNA into cells. Micropipette containing DNA Target cell's nucleus Blank .22 caliber shell Nylon projectile Vent Plate to stop nylon projectile Target cell DNA-coated beads Target cell Suction tube to hold target cell in place Nylon projectile Gene gun 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

Applications of Recombinant DNA Technology Genetic Mapping Locating genes Until 1970, genes identified by labor-intensive methods Simpler and universal methods now available Restriction fragmentation Fluorescent in situ hybridization (FISH)

Figure 8.10 Fluorescent in situ hybridization (FISH).

Figure 8.11 Automated DNA sequencing.

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

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

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

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

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

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 a gene for salt tolerance into tomato and canola plants Transgenic plants survive, produce fruit, and remove salt from soil

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 use to reduce insect damage to crops Gene for Bt toxin inserted into various crop plants Genes for Phytophthora resistance inserted into potato crops

Figure 8.13 Genetically modified papaya plants.

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 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

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

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

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"?