Module 4 Testing Your Own DNA: PCR

To determine whether you have taster or nontaster alleles, you’ll first need a sample of your DNA. This is easily accomplished by a saline mouthwash. You’ll extract the DNA from the cells and make about a billion copies of the region of the TAS2R38 gene that we are interested using a technique called polymerase chain reaction (PCR).

PCR is one of the most important tools in molecular biology, and if you continue in the Biotechnology program you’ll learn many uses and variations of it. PCR relies on the ability of DNA polymerase to synthesize a new strand of DNA complementary to a template strand. DNA polymerase can’t just operate on a single template strand of DNA, though; it is only capable of extending an existing nucleic acid strand. This allows us to use primers, short single strands of DNA that have a sequence of our choice, to determine what region of DNA the polymerase makes a copy of. By altering the temperature, we can denature (separate the DNA strands), anneal (cause primers to bind), and then extend the DNA using DNA polymerase. The figure below shows how PCR works.

PCR occurs over multiple cycles, each containing three steps: denaturation, annealing, and extension. First, double-stranded template DNA containing the target sequence is denatured at approximately 95 °C. The high temperature required to physically, rather than enzymatically, separate the DNA strands is the reason the heat-stable DNA polymerase is required. Next, the temperature is lowered to approximately 50 °C. This allows the DNA primers complementary to the ends of the target sequence to anneal (stick) to the template strands, with one primer annealing to each strand. Finally, the temperature is raised to 72 °C, the optimal temperature for the activity of the heat-stable DNA polymerase, allowing for the addition of nucleotides to the primer using the single-stranded target as a template. Each cycle doubles the number of double-stranded target DNA copies.
The polymerase chain reaction (PCR) is used to produce many copies of a specific sequence of DNA.

 

To understand PCR even better, watch the animation of the process below. Note that the target, the region of DNA that is amplified, is determined by the sequence of the primers you put into the reaction.

Watch

Polymerase Chain Reaction (PCR)