PCR stands for polymerase chain reaction. This process is used to amplify a specific portion of a DNA sample so that it is much more concentrated than the rest of the DNA in the sample. This is done so that that specific segment of DNA can be sequenced and the code can be read and compared to the same segment from other samples. Once this sequence is obtained and analyzed, the genetic differentiation between samples can be found, which is the over all goal of all the lab work I have done. But more about the PCR, in each of the tubes there are several ingredients, and each one must be right, or it is unlikely to be successful. The ingredients are: purified water, dNTP’s (which are the A’s, T’s, C’s, and G’s that DNA is made up of), magnesium (which aids in the bonding process of the dNTP’s), the forward and reverse primers (which ensure that the segment being amplified is the correct one), a very small amount of purified DNA, a buffer to ensure the proper conditions inside the tube while the reaction occurs, and lastly, Taq, a protein from a bacteria found in hot springs in Yellowstone national park. The human body contains a similar protein, which, whenever our cells are dividing, pairs the correct dNTP’s with the correct base on the already existing half strand of DNA as it divides. However, due the high temperatures necessary for the PCR to work in a reasonable amount of time, our version of this protein would be unsuitable. Hence, because the bacteria with a similar protein came from a hot springs, it is capable of withstanding the necessary temperatures. After all of these ingredients are combined in the tubes in the proper quantities, the tubes are then put into a thermocycler, which is a programmable machine, that controls the temperature of the tubes throughout the reaction. For my particular reaction, the temperatures used were: an initial 1 minute at 94°C and then a cycle of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute, this cycle was repeated 32 times and followed by 7 minutes at 72°C. The reason for the temperature changes is that at each temperature, a different step of the process occurs. The step that occurs at 94°C is called denaturation, and this is where the two strands of DNA separate. The next step is is annealing, where the primers find their place on the the half strand of DNA. The final temperature is the extension step, where the Taq starts at the end of the primer and attaches complimentary dNTP’s, and the first time, doesn’t stop until the thermocycler changes the temperature, causing the newly formed copies to split away from the original DNA. When they split, then there is one full copy of each side of the DNA, as well as a partial copy that starts at one end of the region that will be amplified, and goes one way, and one partial copy that starts at the other end, and goes the other way. Then, when the next annealing step occurs, the primers will bond to the partial strands (they will also bond to the full strands, but that doesn’t matter) and go from where the primer started, to the end of the partial strand, which is where the other primer ends. After this first isolation of the region to be amplified has occurred, then the duplication process takes off exponentially, leading to a much higher concentration of the region you are interested in than anything else.
I’ve been learning a lot about lab work these past couple of weeks. I have been working with fin clip tissue samples from Arctic Grayling from across the state of Alaska. I’ve learned how to isolate, and then amplify DNA from these samples in order to have it sequenced and identify genetic variations between specimens. In order to isolate the DNA from a sample, it is first incubated in a solution that breaks down the sample. Next a solution is added to cause the proteins to fall out of solution, and all of this is spun in a centrifuge in order to create a pellet of protein at the bottom of the vial. The remaining liquid, containing the DNA, is poured off into another vial containing isopropanol, which causes the DNA to fall out of solution. This mixture is then once again put into the centrifuge, this time to create a pellet of DNA to form. After this is completed, the isopropanol is poured off, and replaced with a 70% ethanol mixture. The ethanol in the mixture causes the DNA to remain in a pellet, while the water causes it to go back into solution, resulting in a pellet which usually remains attached to the bottom of the vial, but is unstable. The ethanol mixture is then removed with a vacuum leaving only a pellet of isolated DNA which can then be used in later process’. I also recently learned how to amplify certain sections of the DNA, but I don’t quite understand it fully yet, so I’ll be explaining that in the next few days.