PCR AND GEL ELECTROPHORESIS
1. The central purpose of this paper is to describe all of the methods of how PCR was developed and the results of the experiments involving extraction and amplification of DNA (Mullis et. al. 1986).
2. PCR has the ability to isolate specific DNA sequences with the use of primers. This is done by denaturing the DNA (at 95o C) so it is able to anneal to the primers that specify a fragment to be amplified (Mullis et. at. 1986). These primes anneal to a specific sequence of DNA in order to amplify this desired sequence.
Once the annealing process is complete, the primers are extended (at 30o C) by a DNA polymerase added to the reaction (Mullis et. al. 1986). At a certain temperature, the DNA polymerase will add the remaining nucleotides to the DNA strand. These strands of DNA are constantly getting smaller and more specific in sequence until the desired sequence is obtained. This process is repeated until an isolated, amplified sequence of DNA is obtained.
After the third cycle of PCR, only the sequences targeted by the primers should be amplified (Mullis et. al. 1986). In previous cycles, there is only one definite end-the 5’ end, however there is an indefinite end-the 3’ end, which is why the first two cycles do not contain the amplified DNA just yet (Mullis et. al. 1986). By the third cycle, there are two definite ends, which means the targeted sequence has been isolated and amplified (Mullis et. al. 1986).
3. One novel application for PCR is fact that isolating and amplifying specific DNA sequences can help to detect a mutation or sequence that causes a certain disease. PCR allows you to locate this specific sequence that may be causing the disease and locate a disease early on. This ability to locate the disease before you even have symptoms allows doctors to treat a patient even before the disease is detected.
4. As discussed in the Saiki et. al. paper, Klenow fragments are used in enzyme activity. It is used as a tool to make a 5’ overhang
2. PCR has the ability to isolate specific DNA sequences with the use of primers. This is done by denaturing the DNA (at 95o C) so it is able to anneal to the primers that specify a fragment to be amplified (Mullis et. at. 1986). These primes anneal to a specific sequence of DNA in order to amplify this desired sequence.
Once the annealing process is complete, the primers are extended (at 30o C) by a DNA polymerase added to the reaction (Mullis et. al. 1986). At a certain temperature, the DNA polymerase will add the remaining nucleotides to the DNA strand. These strands of DNA are constantly getting smaller and more specific in sequence until the desired sequence is obtained. This process is repeated until an isolated, amplified sequence of DNA is obtained.
After the third cycle of PCR, only the sequences targeted by the primers should be amplified (Mullis et. al. 1986). In previous cycles, there is only one definite end-the 5’ end, however there is an indefinite end-the 3’ end, which is why the first two cycles do not contain the amplified DNA just yet (Mullis et. al. 1986). By the third cycle, there are two definite ends, which means the targeted sequence has been isolated and amplified (Mullis et. al. 1986).
3. One novel application for PCR is fact that isolating and amplifying specific DNA sequences can help to detect a mutation or sequence that causes a certain disease. PCR allows you to locate this specific sequence that may be causing the disease and locate a disease early on. This ability to locate the disease before you even have symptoms allows doctors to treat a patient even before the disease is detected.
4. As discussed in the Saiki et. al. paper, Klenow fragments are used in enzyme activity. It is used as a tool to make a 5’ overhang
References: Mullis, K. et. al. "specific enzymatic amplification of DNA in vitro: the polymerase chain reaction." Cold Spring Harbor Symposia on Quantitative Biology. LI. (1986): 263-273. Print. Saiki, R.K. et. al. "Primer-Directed Enzymatic Amplification of DNA with a Thermostable DNA Polymerase." Science. 239. (1988): 487-491. Print. Zhao, G. et. al. "Realizing directional cloning using sticky ends produced by 3′-5′ exonuclease of Klenow fragment." Indian Academy of Sciences. 38.5 (2013): 857-866. Print.