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.

Key concepts

Denaturation

Denaturation is the first crucial step in the Polymerase Chain Reaction (PCR). During this step, the PCR reaction temperature is elevated to a high range, usually between 92-95°C. The primary purpose of this high temperature is to break the hydrogen bonds that hold the two strands of the DNA double helix together.
This process results in the separation of the double-stranded DNA molecule into two single strands. This "unzipping" of the DNA double helix is essential as it generates the single-stranded DNA templates required for the subsequent stage of PCR. Without denaturation, the DNA strands would remain bound to each other, making it impossible for the following steps to proceed.

• Denaturation temperature: 92-95°C
• Effect: Separation of DNA double-strand into single-strands
• Outcome: Produces templates for the next step

Annealing

Annealing is the second step in PCR, where the reaction temperature is significantly lowered to between 45-65°C. This cooling allows short, specific sequences of DNA, known as primers, to bind or anneal to their complementary sequences on the single-stranded DNA templates.
Primers are vital in PCR as they define the starting point for DNA synthesis. These short sequences are carefully designed to match the target region's start and end points, allowing for the amplification of only the specific DNA segment of interest. The specific temperature at which annealing occurs depends significantly on the characteristics of the primers themselves, such as their length and the sequence of nucleotides they contain.

• Annealing temperature: 45-65°C
• Role of primers: Target specific sequence for amplification
• Adequate annealing is crucial for DNA polymerase activity in the next step

DNA Polymerase

DNA Polymerase plays a pivotal role in the third step of PCR, known as extension or elongation. During this phase, the temperature is increased to a range of 65-75°C, activating a special enzyme called DNA polymerase. Taq polymerase, a commonly used enzyme due to its stability at high temperatures, is a popular choice here.
At this temperature, the DNA polymerase begins synthesis: it adds nucleotides one by one to the 3' end of each primer. It uses the single-stranded DNA as a template to accurately build a new DNA strand from each primer.
The efficiency of this enzyme is what allows PCR to multiply segments of DNA rapidly. Different polymerases might require slightly different temperatures for optimal activity, but for most common polymerases, like Taq, 72°C is optimal.

• Extension temperature: 65-75°C
• Enzymes involved: DNA polymerase, typically Taq polymerase
• Function: Adds nucleotides to form new DNA strands