BME100 s2015:Group2 12pmL4
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LAB 4 WRITE-UP
have the same forward primer and reverse primer
samples will be cross-contaminated
Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, and Extend at 72°C for 30 seconds
Research and Development
PCR - The Underlying Technology
Components and Their Functions in a PCR Reaction
For the PCR Reaction, there are four main components. These components are: Template DNA, Primers, Taq Polymerase, Deoxyribonucleotides (dNTP’s). The template DNA is the target DNA which the PCR process is amplifying. This DNA has a specific sequence that is to be amplified by the PCR process. But before it can be amplified (replicated over and over again), the template strand must first be separated. This is done by adding heat to the system which separates the double-stranded template DNA into individual strands. After they are separated, a primer is used to locate the specific sequence of DNA that is to be amplified. Primers themselves are short segments of DNA that bind to a denatured (separated) strand and signal where the DNA is to start being replicated. Primers have to be selected carefully to ensure that they bind to the site of interest and signal to replicate the section of DNA that follows. After the primer has bound to its respective place on the denatured DNA, Taq Polymerase – an enzyme – comes in and reads the DNA sequence following the primer. The Taq Polymerase matches the template DNA with its respective base pair. In DNA, there are four bases – Adenosine (A), Thymine (T), Cytosine (C), and Guanine (G) – and each bas has its respective partner. A’s bond to T’s and C’s bond to G’s. That being said, the Taq Polymerase matches the Template DNA with its appropriate base pair. These base pairs are free floating in solution in the form of Deoxyribonucleotides. Deoxyribonucleotides are the individual bases – A, T, C, G – and the Taq Polymerase grabs the respective base and pairs it with its proper pair in order to create a two double stranded DNA molecules based upon the original template strand that was placed in solution. This process is repeated over and over again in PCR to create many copies based upon the target sequence of the template DNA.
At the initial step of the PCR reaction, the machine with the samples is brought to 95 degrees Celsius for three minutes. This helps the bonds in the double stranded template DNA start to loosen as the heat is applied. When an additional 30 seconds at 95 degrees Celsius is added, the template DAN completely separates – or Denatures – and creates two single-stranded DNA molecules. DNA does not like to stay split up for long which is why such a high heat is necessary for separating the DNA and why the next step has to follow quickly. In the Annealing step of the PCR process, the primers quickly bind to their respective sites on the two single stranded DNA molecules and this no longer allows the DNA to join back together and form a double helix. Also, this step is carried out for 30 seconds at 57 degrees Celsius. It must have a lower temperature because when the system is at 95 degrees Celsius, nothing can bind due to the high temperatures. The 57 degrees Celsius allows the primer to come in and bind to its appropriate site. In the Extending section, the Taq Polymerase is activated by the change in heat and it locates the newly bound primer and then reads down the DNA and adds the respective Deoxyribonucleotide base pairs to create two double stranded DNA molecules from the original one double stranded DNA molecule. This process is carried out at 72 degrees Celsius for 30 seconds. The Taq Polymerase acts quickly in reading the DNA and adding the appropriate base pairs. When the Taq Polymerase reaches the end of the DNA strand, it falls off (detaches from) the end of the DNA strand and the replication is complete. The Final Step of the PCR cycle is then carried out which hold the temperature at 72 degrees Celsius for 2 minutes and ensures that the bonds are properly formed in the DNA double helix. This concludes one cycle of the PCR machine; however, the same process (initial step through final step) is repeated 34 more times to make a total of 30 PCR reactions which take place during the amplication of DNA via PCR. After the 35 cycles have been carried out, there is one final step that needs to be observed. The system is brought to a final hold at 4 degrees Celsius for a short period of time. This is done to ensure that the DNA will not split apart again by bringing to such a cool temperature where it is not physically possible for the DNA to split apart.
As described earlier, DNA is composed of four pairs of bases: Adenine (A), Thymine (T), Cytosine (C), and Guanine (G). Each Base pairs up with only one other kind of base. This makes it so that the Genetic code is carried out by making sure one base matches with another. For the base pairs, Adenine binds with Thymine and Cytosine binds with Guanine.
In the PCR cycle, base pairing occurs in two places. It first occurs in the Annealing step where primers bind to their respective section on the denatured, single-stranded DNA. This occurs because, as described earlier, primers are short fragments of DNA meaning that since they are a specific sequence, they can only match up with the DNA that holds the base pairs which match up. Base pairing also occurs in the Extending step where Taq Polymerase brings in the Deoxyribonucleotides freely floating in solution and binds them to their respective base pair based upon the original DNA strand.