Question

Each step of the polymerase chain reaction is associated with a particular temperature. Match the temperatures in the left column with the appropriate PCR stage on the right.

A. 94°C
B. 72°C
C. 55°C
D. 94°C
E. 72°C

Answer 1

The PCR steps of denaturation, annealing, and extension correspond to temperatures of 94°C, 55°C, and 72°C, respectively. These temperatures facilitate DNA strand separation, primer annealing, and strand extension by Taq polymerase.

Step-by-step explanation:

The polymerase chain reaction (PCR) involves three temperature-dependent steps: denaturation, annealing, and extension. For denaturation, the double-stranded DNA is separated at a high temperature, typically around 94°C (A and D). During the annealing step, the temperature is lowered to approximately 55°C (C) so that primers can form Watson-Crick base pairs with each strand of the DNA. The extension step occurs at an intermediate temperature, usually about 72°C (B and E), where Taq polymerase extends the DNA strand from the primer. These steps are repeated to exponentially amplify the target DNA fragment.

A standard Polymerase Chain Reaction (PCR) is an in vitro method that allows a single, short region of a DNA molecule (single gene perhaps) to be copied multiple times by Taq Polymerase. From a single copy of DNA (the template), a researcher can create thousands of identical copies using a simple set of reagents and a basic heating and cooling (denaturing and annealing) cycle. The process became automated with the discovery of a heat resistant DNA polymerase from the thermophilic bacterium, Thermus aquaticus (Taq). Taq polymerase can withstand many heating and cooling cycles, which would denature DNA polymerases from other species.

In addition to the template DNA and the Taq polymerase, PCR requires free nucleotides [dNTPs; adenine (A), cytosine (C), guanine (G), thymine (T)] in an equal molar ratio. It also requires two unique single stranded DNA oligonucleotide (oligo) primers, which anneal to the regions upstream (5’) and downstream (3’) of the DNA segment to be amplified. When these reagents are combined in an appropriate buffer, a series of heating (denaturing) and cooling (annealing) steps allow the Taq polymerase to copy the DNA in between the oligo primers. This molecular biology technique creates several micrograms of target DNA from just a few nanograms of template DNA through several cycles of denaturation, annealing, and synthesis. After the PCR is complete, the product can be verified based on size by gel electrophoresis.