Question 1 - Polymerase Chain Reaction Flashcards
Why is it important to use a Taq Polymerase from a thermophilic eubacterial microorganism?
The main reasons that make Thermus aquaticus (Taq) perfect for DNA sequencing are that it’s active across a wide range of temperatures and as such is able to withstand the protein denaturing necessary during PCR so that PCR cycles can be automated, since the polymerase doesn’t need to be added for each cycle.
What is the component that allows PCR to target a specific strand of DNA?
DNA polymerase
Explain the process of PCR (Hint: temperature, template strand, PCR components) - Draw a diagram to explain (10 pts)
is a technique used to amplify a segment of DNA of interest or produce lots and lots of copies.
The PCR machine steps happen in the amplification step. It begins with a segment of a DNA sample placed in a suitable tube along with the reagents and chemicals listed above. The tube is placed into the PCR machine or thermal cycler. The thermal cycler takes the solution through a 3-step process: denaturation, annealing, and extension.
Step 1 - Denaturation
The solution contained in the tube is heated to at least 94°C (201.2°F) using a thermal cycler. The heat breaks the hydrogen bonds of the original DNA sample and separates the DNA into single strands (this is termed denaturation of double-stranded DNA).
Step 2 - Anneling
The sample mixture is then cooled to between 50 to 60°C (122 to 140°F) allowing the DNA primers and the DNA polymerase enzyme to bind to the individual strands of DNA that were separated by the heat (this is termed annealing of the primers). At this point, the nucleotides (A, T, C, G) from the added mixture solution will pair with the individual separated strands of DNA that resulted from the heating process.
Step 3 - Extension
Once joined together, they form a new complementary strand of DNA (termed extension of the DNA). Thus, a new duplicate double-stranded DNA molecule has been formed from each of the single strands of the original sample molecule. The temperature cycles from 95°C to 50 to 60°C. The cycle is then repeated about 35 to 40 times using the thermal cycler which automatically repeats the heating and cooling cycles of the process. Resulting DNA sequence is doubled each time the heating/cooling cycle is conducted by the cycler. Thus, what started out as a single short segment of DNA from one sample can be amplified to form millions of copies after 35 doubling cycle
Step 4 - Analysis with Electrophoresis
Once the PCR process is completed, the resulting amplified (replicated) segments generated can then be compared to other nucleotide segments from a known source. The PCR-generated nucleotide sequences are then placed next to known nucleotide sequences from humans, pathogens, or other sources in a separating gel. Electrical current is then run through the gel, and the various nucleotide sequences within the gel form bands that resemble a ladder, according to their electrical charge and molecular size. This is termed gel electrophoresis. Bands or ladder-like steps that migrate to the same levels in the gel show identity of nucleotide sequences. This method is one of the most popular ways PCR tests are completed.
