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The Polymerase Chain Reaction

Bio Project

Kyle Hogan

on 8 December 2012

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Transcript of The Polymerase Chain Reaction

Polymerase Chain Reactions The process of the polymerase chain reaction uses a thermocycler (heat-controlling machine) and taq DNA polymerase (an added non-native polymerase) to make the process of DNA denaturing and synthesis more efficient. Through this method, DNA can be synthesized at a rate of approximately one million copies in two hours! (Cold Spring Harbor Laboratory, 2012) This is much faster than the natural process. Using this technology allows many medical advancements to be made in the genetics field. What it is... How DNA is Used... There are a few ethical concerns connected to PCR. The largest ethical issue that surrounds the use of PCR technology has to do with the testing of amniotic fluid of in utero organisms (Ragnar, 2012). Many people believe that to take samples of cells from organisms which only contain eight cells is unethical in that it may be a damaging procedure to the embryo (Edvotek, 2012). Another source of ethical concern in regards to PCR stems from the results of the potential amniocentesis. When the DNA is acquired from the embryo, it can be replicated and used to scan for potential birth defects or diseases. This will always be a sore spot among then people who believe this knowledge will lead to drastic meausres such as aborting a child with down's syndrome (Otto, 1997). Ethical Issuses... The polymerase chain reaction is relatively new technology. The idea was conceived in 1983 by Dr. Kary Mullis, who was working for Cetus Corporation. They found that heating the DNA helped release the hydrogen bonds, thus releasing the double helix and speeding up the replication process (Roche Molecular Diagnosotics, 2012). However, they also found that the polymerase that helped form the new strands would denature at the high temperatures. This is what lead to the use of the artificial taq DNA polymerase. He and the other scientists at the Cetus developed the idea until it was highly functional and then patended it in 1985. This is also the year that the first publications came out describing the technology and its applications to clinical diagnosis (Roche Molecular DIagnostics). History of PCR... Technology of the Future Ethical Solutions... 1. The DNA is denatured at 94-96 degrees Celcius 2. The primers anneal (3' to 5') to the specified genome to be copied 3. Taq DNA prymase binds to the 3' end of the primers and synthesizes the new strand (5' to 3') Cycle 1 3' 5' 3' 5' 5' 3' 3' 5' 94-96 degrees Celsius 55-65 degrees Celsius 72 degrees Celsius (Cold Spring Harbor Laboratory, 2012) Cycle 2 4.Steps 1-3 are repeated to result in four copies of the target genome sequence Cycle 3 5. Repeat steps 1-3 with the final product of cycle 2. Two fragments of unattached target genome are the final product, along with six fragments of the target genome attached to extraneous DNA Unattached target genome: Target genome + extraneous DNA 6. These cycles repeat for a few hours, or until the desired amount of target genomes have been produced (Cold Spring Harbor Laboratory, 2012) (Cold Spring Harbor Laboratory, 2012) (ayalush78, 2007) (ayalush78, 2007) Principles The thermocycler alternates temperatures every few minutes to allow the process to work more effectively. This is because at higher temperatures, DNA's hydrogen bonds will become less stable, causing the DNA to split into two segments. This occurs between 94-96 degrees Celsius. Another temperature that the thermocycler uses is between 50-65 degrees Celsius, which is the ideal temperature for primers to anneal to the base pairs. Lastly in the cylce, the thermocycler heats up to a temperature suitable for the taq primase to synthesize the new strand of DNA (Cold Spring Harbor Laboratory, 2012). The cycles are repeated inside of the thermocycler for a few hours, or until the desired amount of target genomes are produced (Rice, 2012).
Since the temperature is being raised to levels that are higher than DNA (of humans and most other organisms) can replicate at, a supplementary polymerase was needed because the DNA polymerase that was already present would denature. Taq polymerase is added because it will not Thermocyler Continue to taq polymerase... Continue to thermocyclers... taq DNA Polymerase denature at the higher temperatures (Bowen, 1999) and therefore allows for the process of DNA replication to occur,even after it has passed extreme temperatures. This enzyme originates from a hot springs bacterium called Thermus Aquaticus (Bowen, 1999). It has since been commercialized for use in the PCR (polymerase chain reaction) process. Even though there are some concerns with the ethical values of PCR, there are far more potential benefits. These include disease research advancement, blood screening and genetic testing (Roche Molecular Diagnostics, 2012). With the application of PCR, a lot of progress has been made towards the study of infectious disease like HIV, HPV and tuberculosis (Roche Molecular Diagnostics, 2012). By having the ability to replicate the genomes involved with the diseases, researchers can more effectively study it without worrying about financing extensive amounts of test DNA. This means that research into these areas is not as cost prohibitive as it once was. Blood screening has also been improved with the use of PCR. This is due to the increased speed with which testable blood is available. Now, the DNA of the blood being tested can be quickly replicated rather than having to grow the samples in bacteria culture artificially. Lastly, PCR is used to provide safe genetic screening. Despite potential ethical concerns with genetic screening, PCR provides a method of safe screening by requiring only a very small sample in order to provide necessary volumes of DNA for testing. This has advanced the ability for doctors to diagnose genetic disorders in embryos. References
ayalush78 (Director). (2007). PCR [Motion Picture].
Bowen, R. (1999, December 27). Thermostable DNA Polymerases. Retrieved from Restriction Endonucleases and DNA Modifying Enzymes: http://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/hotpolys.html
Cold Spring Harbor Laboratory. (2012). Polymerase Chain Reaction. Retrieved from DNA Learning Center: http://www.dnalc.org/resources/animations/pcr.html
Edvotek Inc. (2012). Polymerase Chain Reaction (PCR). Retrieved from Edvotek: http://www.edvotek.com/PCR
Levi, R. (2012). Genetic Diagnosis by PCR. Retrieved from SBU evaluates health care technologies: http://www.sbu.se/sv/Publicerat/Gul/1860/
National Human Genome Reasearch Institute. (2012, February 27). Polymerase Chain Reaction (PCR). Retrieved from National Human Genome Research Institute: National Institutes of Health: http://www.genome.gov/10000207
Otto, C. (1997). Ethical Issues of Genetic Screening. Retrieved from Ethical Issues of Genetic Screening: http://www.ndsu.edu/pubweb/~mcclean/plsc431/students/otto.html
Rice, G. (2012, November 27). Polymerase Chain Reaction. Retrieved from Microbial Life: http://serc.carleton.edu/microbelife/research_methods/genomics/pcr.html
Roche Molecular Diagnostics. (2012, November 10). Applications of PCR. Retrieved from Roche: http://molecular.roche.com/About/pcr/Pages/ApplicationsofPCR.aspx
Roche Molecular Diagnostics. (2012). Timeline of PCR and Roche. Retrieved from Roche Molecualr Diagnostics: http://molecular.roche.com/About/pcr/Pages/PCRTimeline.aspx

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