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Biotechnology Chapter 17
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DNA Manipulation The molecular biology revolution started with the discovery of restriction endonucleases -Enzymes that cleave DNA at specific sites These enzymes are significant in two ways 1. Allow a form of physical mapping that was previously impossible 2. Allow the creation of recombinant DNA molecules (from two different sources)
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DNA Manipulation Restriction enzymes recognize DNA sequences termed restriction sites There are two types of restriction enzymes: -Type I = Cut near the restriction site -Rarely used in DNA manipulation -Type II = Cut at the restriction site -The sites are palindromes -Both strands have same sequence when read 5’ to 3’
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DNA Manipulation Type II enzymes produce staggered cuts that generate “sticky ends” -Overhanging complementary ends Therefore, fragments cut by the same enzyme can be paired DNA ligase can join the two fragments forming a stable DNA molecule
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Recombinant DNA molecule Restriction endonuclease cleaves the DNA
G T A DNA ligase joins the strands. DNA duplex Sticky ends Restriction sites EcoRI Recombinant DNA molecule Restriction endonuclease cleaves the DNA DNA from another source cut with the same restriction endonuclease is added. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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Gel Electrophoresis A technique used to separate DNA fragments by size
The gel (agarose or polyacrylamide) is subjected to an electrical field The DNA, which is negatively-charged, migrates towards the positive pole -The larger the DNA fragment, the slower it will move through the gel matrix DNA is visualized using fluorescent dyes
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Restriction Enzyme Digestion Gel Electrophoresis
Restriction endonuclease 1 cut site 2 cut site Reaction 1 2 3 Restriction endonuclease 3 Short segment Long segment Medium segment Mixture of DNA fragments of different sizes in solution placed at the top of “lanes” in the gel Gel Lane Anode + Cathode - Power source Reaction 2 Reaction 1 Reaction 3 Shorter fragments Longer Visualizing Stained Gel Gel is stained with a dye to allow the fragments to be visualized. DNA samples are cut with restriction enzymes in three different reactions producing different patterns of fragments Samples from the restriction enzyme digests are introduced into the gel. Electric current is applied causing fragments to migrate through the gel. Restriction Enzyme Digestion Gel Electrophoresis a. b. c. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Buffer
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Transformation Transformation is the introduction of DNA from an outside source into a cell Natural transformation occurs in many species -However, not in E. coli, which is used routinely in molecular biology labs -Artificial transformation techniques have been developed to introduce foreign DNA into it
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Molecular Cloning A clone refers to a genetically identical copy
Molecular cloning is the isolation of a specific DNA sequence (usually protein-encoding) -Sometimes called gene cloning The most flexible and common host for cloning is E. coli Propagation of DNA in a host cell requires a vector
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Vectors Plasmids are small, circular extrachromosomal DNA molecules
-Used for cloning small pieces of DNA -Have three important components 1. Origin of replication 2. Selectable marker 3. Multiple cloning site (MCS)
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Vectors
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Vectors Phage vectors are modified bacterial viruses
-Most based on phage lambda (l) of E. coli -Used to clone inserts up to 40 Kbp -Have two features not shared with plasmid vectors -They kill their host cells -They have linear genomes -Middle replaced with inserted DNA
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Vectors
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Vectors Artificial chromosomes -Used to clone very large DNA fragments
-Bacterial artificial chromosomes (BACs) -Yeast artificial chromosomes (YACs)
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DNA Libraries A collection of DNA fragments from a specific source that has been inserted into host cells A genomic library represents the entire genome A cDNA library represents only the expressed part of the genome -Complementary DNA (cDNA) is synthesized from isolated mRNA using the enzyme reverse transcriptase
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Eukaryotic DNA template exons introns 1 2 3 4
5´ cap Eukaryotic DNA template exons introns 1 2 3 4 Transcription Primary RNA transcript Mature RNA transcript 3´ poly-A tail Degraded mRNA mRNA–cDNA hybrid Isolation of mRNA Addition of reverse transcriptase Addition of mRNA- degrading enzymes DNA polymerase Double-stranded cDNA with no introns Reverse utilizes mRNA to create cDNA. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Introns are cut out, and coding regions are spliced together.
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DNA Libraries Molecular hybridization is a technique used to identify specific DNAs in complex mixtures -A known single-stranded DNA or RNA is labeled -It is then used as a probe to identify its complement via specific base-pairing -Also termed annealing
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DNA Libraries Molecular hybridization is the most common way of identifying a clone in a DNA library -This process involves three steps: 1. Plating the library 2. Replicating the library 3. Screening the library
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3. The filter is washed with a solution to break the cells
Film Filter paper 1. Colonies of plasmid containing bacteria, each containing a single DNA from the library, are grown on agar. 3. The filter is washed with a solution to break the cells open and denature the DNA, which sticks to the filter at the site of each colony. The filter is incubated with a radioactively labeled probe that can form hybrids with complementary DNA in the gene of interest. 4. The only sites on the filter that will retain probe DNA will contain DNA complementary to the probe. These represent the sites of colonies containing the gene of interest. 5. A comparison with the original plate identifies the colony containing the 2. A replica of the plate is made by pressing a piece of filter paper against the agar and bacterial colonies. Some cells from each colony adhere to the filter.
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DNA Analysis Restriction maps
-Molecular biologists need maps to analyze and compare cloned DNAs -The first maps were restriction maps -Initially, they were created by enzyme digestion & analysis of resulting patterns -Many are now generated by computer searches for cleavage sites
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DNA Analysis Southern blotting
-A sample DNA is digested by restriction enzymes & separated by gel electrophoresis -Gel is transferred (“blotted”) onto a nitrocellulose filter -Then hybridized with a cloned, radioactively-labeled DNA probe -Complementary sequences are revealed by autoradiography
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4. Nitrocellulose with bound DNA is incubated with radioactively
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 3. DNA in the gel is transferred, or “blotted,” onto the nitrocellulose. 4. Nitrocellulose with bound DNA is incubated with radioactively labeled nucleic acids and is then rinsed. 5. Photographic film is laid over the filter and is exposed only in areas that contain radioactivity (autoradiography). Bands on the film represent DNA in the gel that is complementary to the probe sequence. Size markers Hybridized nucleic acids Film Gel Buffer Sponge Nitrocellulose filter 1. Electrophoresis is performed, using radioactively labeled markers as a size guide in the first lane. Test nucleic Radioactively labeled markers with specific sizes Electrophoretic gel Electrophoresis DNA fragments within bands Radioactive probe (single- stranded DNA) 2. The gel is covered with a sheet of nitrocellulose and placed in a tray of buffer on top of a sponge. Alkaline chemicals in the buffer denature the DNA into single strands. The buffer wicks its way up through the gel and nitrocellulose into a stack of paper towels placed on top of the Stack of paper towels —TTACC— —AATGG— Sealed container Nitrocellulose paper now contains nucleic acid “print”
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DNA Analysis Northern blotting
-mRNA is electrophoresed and then blotted onto the filter Western blotting -Proteins are electrophoresed and then blotted onto the filter -Detection requires an antibody that can bind to one protein
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DNA Analysis RFLP analysis
-Restriction fragment length polymorphisms (RFLPs) are generated by point mutations or sequence duplications -These fragments are often not identical in different individuals -Can be detected by Southern blotting
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c. Gel electrophoresis of Original Sequence of Restriction Sites
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. a. Three different DNA duplexes b. Cut DNA c. Gel electrophoresis of restriction fragments Original Sequence of Restriction Sites (no mutations) Point Mutations Change the Sequence of Restriction Sites Sequence Repetitions Can Occur Between Larger fragments Smaller restriction enzyme cutting sites Sequence duplication Single base-pair change + –
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DNA Analysis DNA fingerprinting
-An identification technique used to detect differences in the DNA of individuals -Makes use of a variety of molecular procedures, including RFLP analysis -First used in a US criminal trial in 1987 -Tommie Lee Andrews was found guilty of rape
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DNA Analysis
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DNA Analysis DNA sequencing -A set of nested fragments is generated
-End with known base -Separated by high-resolution gel electrophoresis, resulting in a “ladder” -Sequence is read from the bottom up
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DNA Analysis DNA sequencing
-The enzymatic method was developed by Frederick Sanger -Dideoxynucleotides are used as chain terminators in DNA synthesis reactions
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Manual Enzymatic DNA Sequencing
5´ 3´ Reaction for ddG Primer Template DNA polymerase for ddC for ddA for ddT Longer segments Shorter + –
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DNA Analysis DNA sequencing
-The enzymatic technique is powerful but is labor intensive and time-consuming -The development of automated techniques made sequencing faster and more practical -Fluorescent dyes are used instead of radioactive labels -Reaction is done in one tube -Data are assembled by a computer
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Manual Enzymatic DNA Sequencing Automated Enzymatic DNA Sequencing
5´ 3´ Primer Template DNA polymerase C A T Manual Enzymatic DNA Sequencing Automated Enzymatic DNA Sequencing a. b. Reaction for ddG for ddC for ddA for ddT Longer segments Shorter Laser Photo detector reads colors + – Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
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DNA Analysis Polymerase chain reaction (PCR) -Developed by Kary Mullis
-Allows the amplification of a small DNA fragment using primers that flank the region -Each PCR cycle involves three steps: 1. Denaturation (high temperature) 2. Annealing of primers (low temperature) 3. DNA synthesis (intermediate temperature) -Taq polymerase
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DNA is denatured into single strands 5´ 3´ Primers anneal to DNA Taq DNA polymerase PCR machine Cycle 2: 4 copies Cycle 3: 8 copies 1. Sample is first heated to denature DNA. 2. DNA is cooled to a lower temperature to allow annealing of primers. 3. DNA is heated to 72°C, the optimal temperature for Taq DNA polymerase to extend primers. DNA segment to be amplified After 20 cycles, a single fragment produces over one million (220) copies!
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DNA Analysis Polymerase chain reaction (PCR)
-Has revolutionized science and medicine because it allows the investigation of minute samples of DNA -Forensics -Detection of genetic defects in embryos -Analysis of mitochondrial DNA from early human species
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DNA Analysis Yeast two-hybrid system
-Used to study protein-protein interactions -Gal4 is a transcriptional activator with a modular structure -The Gal4 gene is split into two vectors -Bait vector: has DNA-binding domain -Prey vector: has transcription-activating domain -Neither of these alone can activate transcription
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DNA Analysis Yeast two-hybrid system
-When other genes are inserted into these vectors, they produce fusion proteins -Contain part of Gal4 and the protein of interest -If the proteins being tested interact, Gal4 function will be restored -A reporter gene will be expressed -Detected by an enzyme assay
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Fusion proteins DNA- binding domain
Bait vector Bait protein Prey vector Prey protein Reporter gene Yeast nucleus Yeast cell Inserted DNA Gal4 protein RNA polymerase DNA DNA- binding domain Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Transcription- activating domain
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Genetic Engineering Has generated excitement and controversy
Expression vectors contain the sequences necessary to express inserted DNA in a specific cell type Transgenic animals contain genes that have been inserted without the use of conventional breeding
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Genetic Engineering In vitro mutagenesis
-Ability to create mutations at any site in a cloned gene -Has been used to produce knockout mice, in which a known gene is inactivated -The effect of loss of this function is then assessed on the entire organism -An example of reverse genetics
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Medical Applications Human proteins
-Medically important proteins can be produced in bacteria -Human insulin -Interferon -Atrial peptides -Tissue plasminogen activator -Human growth hormone
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Medical Applications
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Medical Applications Vaccines
-Subunit vaccines: Genes encoding a part of the protein coat are spliced into a fragment of the vaccinia (cowpox) genome -DNA vaccines: Depend on the cellular immune response (not antibodies)
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Medical Applications 2. Herpes simplex 3. Vaccinia DNA
Human immune response Gene specifying herpes simplex surface protein Harmless vaccinia (cowpox) virus Herpes simplex virus 2. Herpes simplex gene is isolated. 3. Vaccinia DNA is extracted and cleaved. 4. Fragment containing surface gene combines with cleaved vaccinia DNA. 5. Harmless engineered virus (the vaccine) with surface like herpes simplex is injected into the human body. 6. Antibodies directed against herpes simplex viral coat are made. 1. DNA is extracted.
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Medical Applications Gene therapy
-Adding a functional copy of a gene to correct a hereditary disorder -Severe combined immunodeficiency disease (SCID) illustrates both the potential and the problems -Successful at first, but then patients developed a rare leukemia
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Agricultural Applications
Ti (tumor-inducing) plasmid is the most used vector for plant genetic engineering -Obtained from Agrobacterium tumefaciens, which normally infects broadleaf plants -However, bacterium does not infect cereals such as corn, rice and wheat
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Agricultural Applications
Gene of interest Agrobacterium Plasmid Plant nucleus 1. Plasmid is removed and cut open with restriction endonuclease. 2. A gene of interest is isolated from the DNA of another organism and inserted into the plasmid. The plasmid is put back into the Agrobacterium. 3. When used to infect plant cells, Agrobacterium duplicates part of the plasmid and transfers the new gene into a chromosome of the plant cell. 4. The plant cell divides, and each daughter cell receives the new gene. These cultured cells can be used to grow a new plant with the introduced gene.
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Agricultural Applications
Gene guns -Uses bombardment with tiny gold particles coated with DNA -Possible for any species -However, the copy number of inserted genes cannot be controlled
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Agricultural Applications
Herbicide resistance -Broadleaf plants have been engineered to be resistant to the herbicide glyphosate -This allows for no-till planting
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Agricultural Applications
Pest resistance -Insecticidal proteins have been transferred into crop plants to make them pest-resistant -Bt toxin from Bacillus thuringiensis Golden rice -Rice that has been genetically modified to produce b-carotene (provitamin A) -Converted in the body to vitamin A
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Agricultural Applications
Daffodil phytoene synthase gene (psy) psy crtI lcy Phytoene Carotene desaturase -Cyclase Genes introduced into rice genome Expression in endosperm GGPP Lycopene -Carotene (Provitamin A) Bacterial carotene gene (crtI) lycopene -cyclase gene (lcy) Rice chromosome
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Agricultural Applications
Adoption of genetically modified (GM) crops has been resisted in some areas because of questions about: -Crop safety for human consumption -Movement of genes into wild relatives -Loss of biodiversity
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Agricultural Applications
Biopharming -Transgenic plants are used to produce pharmaceuticals -Human serum albumin -Recombinant subunit vaccines -Against Norwalk and rabies viruses -Recombinant monoclonal antibodies -Against tooth decay-causing bacteria
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Agricultural Applications
Transgenic animal technology has not been as successful as that in plants -One interesting example is the EnviroPig -Engineered to carry the gene for the enzyme phytase -Breaks down phosphorus in feed -Reduces excretion of harmful phosphates in the environment
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Agricultural Applications
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