Question

In a typical PCR reaction, describe what is happening in stages occurring at temperature ranges (a) \(92-95^{\circ} \mathrm{C},\) (b) \(45-65^{\circ} \mathrm{C},\) and (c) \(65-75^{\circ} \mathrm{C}\)

Short answer

Question: Describe the events that occur in a polymerase chain reaction (PCR) at the following temperature ranges: (a) 92-95°C, (b) 45-65°C, and (c) 65-75°C. Answer: In PCR, (a) 92-95°C corresponds to the denaturation step, where double-stranded DNA separates into single strands. (b) 45-65°C is the annealing step, during which primers bind to complementary sequences on the single-stranded DNA templates. (c) 65-75°C is the extension step, at which DNA polymerase synthesizes new DNA strands using the original templates.

Step-by-step solution

Denaturation (92-95°C)

In the denaturation step, the temperature is raised to between 92-95°C. This high temperature causes the double-stranded DNA molecule to separate into two single strands. The hydrogen bonds between the complementary base pairs of the DNA strands are broken, and the DNA helix "unzips" as a result. This creates the templates necessary for the next step, annealing.

Annealing (45-65°C)

In the annealing step, the temperature is lowered to a range of 45-65°C. During this stage, short DNA sequences called primers anneal, or bind, to their complementary sequences on the single-stranded DNA templates. These primers are designed to flank the target DNA sequence, allowing the amplification of only the desired region. The specific annealing temperature depends on the primers' length and sequence composition to ensure accuracy and specificity.

Extension (65-75°C)

In the extension step, the temperature is raised again, this time to a range of 65-75°C. At this temperature, a heat-stable DNA polymerase enzyme, such as Taq polymerase, becomes active. The polymerase starts at the 3' end of the annealed primers and adds complementary nucleotides to synthesize new DNA strands, using the original single-stranded DNA templates as a guide. The extension temperature depends on the DNA polymerase used; for example, 72°C is optimal for Taq polymerase. These three steps are repeated for several cycles (typically 25-40 cycles) to generate a large number of copies of the target DNA sequence. PCR is a powerful molecular biology tool with various applications, such as DNA cloning, gene expression analysis, and DNA fingerprinting.