Bio 102 Assignment 3: Part 1
1. What is the purpose of PCR?
The purpose of PCR, polymerase chain reaction, is to amplify small segments of DNA for analyses. To amplify a DNA barcode region, you have to choose the appropriate set of primers, which depends on your organism, for each sample. Essentially, studying isolated pieces of DNA without PCR is nearly impossible.
2. What are the major components of a PCR reaction?
The major components of a PCR reaction are the DNA template, which is a sample of DNA that contains the target sequence, and the DNA polymerase, which are the strands that will get separated from each other after high temperature is applied to the DNA.
3. What is the purpose of heating the DNA sample to 94-96°C?
The high temperature causes the hydrogen bonds between the bases in the DNA sample to break-allowing the two strands to separate. The temperature needs to be maintained long enough that the DNA strands separate, and it usually takes between 15-30 seconds. This is known as the denaturing stage.
4. Why must the polymerase be from Thermophillus aquaticus (Taq) or another extremophile bacteria?
The PCR must be from Taq or another extremophile bacteria because they can tolerate high temperatures and are very heat resistant, which is fundamental to PCR reactions. Thermophiles, specifically Taq, grow best at sites with temperatures ranging from 70-100°C, which is perfect for the denaturing stage where DNA polymerase is isolated. This is why the polymerase needs to contain Taq or another extremophile bacteria .
5. What do we call the primers sticking to the DNA template? At what temperature does this happen (how does length/composition of primers affect it)?
The primers that stick to the DNA template are called universal primers. This happens at temperatures ranging from 45-60°C, or the annealing stage. The temperature is directly affected by the length/composition of the primers. The optimal length is said to be 18-22 base pairs because it’s long enough for adequate specificity and short enough for them to bind easily to the template .
6. What are some applications of PCR?
There are many applications of PCR. It can be used to amplify certain genes associated with a disorder from the DNA of patients-making it valuable in the field of genetic testing. It can also be used to test for bacteria or DNA virus in a patient’s body, and the amplification of the pathogen if it is present. In the field of forensics, you may receive a sample of DNA from someone’s hair left at a crime scene, as well as the DNA of a few possible suspects. You would need to perform PCR on the DNA samples to figure out which suspects’ DNA matches the DNA sample left at the crime scene