Genetic Engineering and Biotechnology (3.5)
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Notes:Biotechnology PPT - Notes
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Polymerase Reaction Chain
The Polymerase Chain Reaction (PCR) was first invented in 1983 by scientist Kary Mullis (Nobel Prize winner 1993).
PCR is a SYNTHETIC model of DNA replication, that occurs in a test tube. It's purpose is to amplify a section or sequence of DNA in order for us to be able to see it. Ingredients necessary to make this function are similar to DNA replication.
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Inside the Thermocycler:
Virtual Lab- try your hand at the procedure
PCR procedure and graph
- Heat to 95ºC (Denature)
- DNA denatures into its two strands-Symbolizing Helicase in DNA replication
- Why use heat instead of enzymes to denature it? Enzyme are heat sensitive and will be denatured at such high temperatures, which would require replacing the enzymes every round, not very cost efficient method.
- Cool down to 58ºC- 65°C (Annealing)
- Primer sequences stick to the template as the temperature decreases
- Heat to 72ºC (Synthesis/ Extension)
- Taq polymerase attach at the primer and make a complimentary copy of the DNA (See below on Taq Polymerase)
- Repeat process roughly 30-40 times, giving us millions of copies of the same DNA sequence.
Virtual Lab- try your hand at the procedure
PCR procedure and graph
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Taq Polymerase
Taq Polymerase is the DNA Polymerase III found in the bacteria Thermus aquaticus, which lives in the Hot Springs of Yellowstone National Park. The Hot Springs have a normal temperature of 95°C. When the discovery of this bacteria was first made it initially had no importance, and was simply filed away into the archieves of science. 30 years later, the reserach was rediscovered and used to develop the PCR method.
Kary Mullis, who developed PCR, received the Nobel Prize in Chemistry in 1993 for his work on PCR.
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Restriction Enzymes
Restriction enzymes (RE) are found in bacteria cells. They are used to cut DNA sequences at very specific sequences. See the various types and their cutting marks on the left.
Restriction enzymes can cut DNA sequences in either Blunt or Sticky ends.
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Gel Electrophoresis
Gel Electrophoresis follows PCR. It is visual way to see the pieces of DNA we just amplified. It uses electrical impulses to pull the DNA through a matrix like jello (called Agarose). DNA in negatively charged, so it moves towards a positive pole and away from the negative pole and the matrix causes longer pieces to move slower than smaller pieces.
Gel Electrophoresis is used for:
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How it works?
Once the DNA is amplified, it can either be put into the Gel electrophoresis machine direction OR cut with Restriction enzymes.
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REstriction Enzymes & Gel Electrophoresis
When the same sequence of DNA is cut with different Restriction Enzymes you end up with different banding patterns. The cuts made create different lengths of DNA, which show up on a gel differently due to size. Depending on the sequence, the number of cuts can vary.
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Virtual Lab- Utah, good overview of the process
Demand a Verdict- DNA Profiling Restriction enzymes + Gel Electrophoresis |
Tandem Repeat DNA
Tandem Repeated DNA are short sequences that are repeated over and over. Every person has a different number of the same repeated sequences. These repeated sequences increase or decrease the length of pieces of DNA
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When the DNA is cut with the same Restriction Enzyme, Tandem Repeated DNA helps to give each person a unique DNA Fingerprint through Gel Electrophoresis, because the length of DNA pieces increase or decrease based on these repeats. The more repeats the longer the piece of DNA and the slower the piece moves through the Gel.
Some individuals will have the same number of repeats, so using only one Restriction Enzyme will sometimes yield the same results. So forensic scientists often use multiple restriction enzymes on different pieces of DNA to ensure that each DNA profile is unique to that individual.
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DNA Profiling
DNA Profiling is the combination of PCR, Restriction Enzymes, Tandem Repeats, and Gel Electrophoresis. When put all together it can be used to create a "map" of a person's DNA that tells one person apart from another. A person's DNA profile is similar to a person's fingerprints. It is unique to the person. As a result it can be used for crime scene investigations where blood/ cells are found. It can also be used in paternity and maternity testing. Anthropology and Evolutionary Biology use it to identify inheritance and ancestry of various groups in how closely related they are.
Take the gel electrophoresis done on this crime scene to the left. The "Crime Scene" blood comes up with a specific banding pattern. The three suspects also have their DNA tested, clearly suspect two was at the crime scene... now whether they did it or not, that is another story.
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In the case of paternity testing, like the one on the right, we are looking for cross markers.
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Genetically Modified Organisms
Genetic Modified Organisms (GMOs) are organism that have had genes transferred to them that are not originally theirs.
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How GMOs are made
GMOs are made usually using restriction enzymes and bacterial plasmids (small pieces of circular DNA).
- Plasmids are removed and cut with restriction enzymes (sticky ends)
- mRNA of desired gene gathered and “reverse transcriptase” to make DNA
- cut with same restriction enzyme
- Two are put together- to insert the DNA into the plasmid
- DNA ligase is used to seal the ends
- Tthe plasmid is the inserted into a bacteria cell (ex. E. coli)
- Bacteria are then cultured to produce what it is suppose to produce
- Bacteria is used to insert the foreign gene into the chromosome of a larger organism (plant or animal)
- Plant/ Animal cells are cultured and the organism grown
Genetically Modified Organism Examples
Human InSulin
Insulin is a hormone produced by the pancreas which allows the body cells to uptake glucose
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Bt Corn
Bt Corn was genetically modified to produce a protein toxin (Bt) that is lethal to the European corn borer, an insect that significatly affects the healthy and yield of corn crops.
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Bt Corn was introduced as a way to increase crop yield, and has be largely successful.
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The GMO Debate
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Genetically Modified Organisms- overview + examples
On the Other side of the story- non-GMO Project GMOs- World Health Organisation Pros and Cons of GMOs- Environmental Science Pros and Cons of GMOs- Energy Conservation |
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Pros
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Cons
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Cloning
A clone is a group of genetically identical organisms or a group of genetically identical cells derived from single parent cell.
Plants are easily cloned from roots, stems or leaves. However, animals can only be cloned from early stages of development (blastocyst). Identical twins are naturally occuring clones. Yet, we can not predict when twins will occur, therefore animals with desirable traits are cloned to continue passing them on. The first successfully cloned mammal was Dolly the sheep (1996). Dolly the sheep |
How did they clone Dolly?
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Types of Cloning
Two types of cloning:
Pros and Cons of Therapeutic Cloning |
Pros and Cons of Therapeutic Cloning
Pros:
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Cons:
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Human Genome Project
A Genome is the whole of the genetic information of an organisms (all ATCGs in order on the various chromosomes in the organisms).
In 2003 the first human genome was sequenced. This was made possible by the development of the capillary electrolysis sequencing method, based off of Fredrick Sanger's Chain Termination Method (1977). Sanger's Method:
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HGP was an international research effort to sequence the genome of a human, completed in 2003
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Overview of the Human Genome Project
Yesterday, Today, Tomorrow- HGP facts about various factors Human Genome Resources- NCBI databases |
Just the Human Genome Project alone is the Full Employment Act for bioethicists.
— Arthur L(eonard) Caplan
Quoted in Michael Schrage, 'Increasing Medical Dilemmas Mean Job Security for Budding Bioethicists', San Jose Mercury News (13 Oct 1992), 3D.