Basic Molecular Biology: PCR and Real-Time PCR – Principle of PCR

Summary

Polymerase chain reaction (PCR) is a technique used to amplify specific DNA fragments. It involves three main steps: denaturation, annealing, and extension. In denaturation, heat separates the DNA into single strands. During annealing, primers bind to complementary sequences on the DNA at a precise temperature. In extension, DNA polymerase adds nucleotides to the primer’s 3’ end, creating new DNA strands. These steps are repeated multiple times to produce many copies of the DNA segment. The success of PCR depends on careful temperature control and primer selection, ensuring specific and efficient DNA amplification.

Takeaways

• 😀 PCR (Polymerase Chain Reaction) is a technique used to amplify specific DNA fragments from a DNA template.
• 😀 PCR consists of three basic steps: denaturation, annealing, and extension.
• 😀 Denaturation is the first step, where heat is applied to separate double-stranded DNA into two single strands.
• 😀 Annealing follows denaturation, where the temperature is reduced to allow primers to bind to complementary sequences on the DNA templates.
• 😀 The annealing temperature depends on the melting temperature (Tm) of the primers, and it should be carefully selected.
• 😀 The optimal annealing temperature is typically 3–5°C below the melting temperature of the primers.
• 😀 If the annealing temperature is too high, primer binding is hindered, while too low a temperature can cause non-specific binding.
• 😀 Extension occurs after primer hybridization, where DNA polymerase adds nucleotides to the 3' ends of the primers.
• 😀 The extension step temperature is optimized for the best performance of DNA polymerase.
• 😀 The length of the extension step depends on the polymerase’s extension rate and the desired length of the PCR product.
• 😀 One complete cycle of PCR involves denaturation, annealing, and extension, and this cycle is repeated multiple times to generate large amounts of the targeted DNA sequence.

Q & A

What is the main purpose of PCR?

The main purpose of PCR (Polymerase Chain Reaction) is to amplify specific DNA fragments from a DNA template, allowing researchers to generate large quantities of a particular DNA sequence.

What are the three basic steps in the PCR process?

The three basic steps in the PCR process are denaturation, annealing, and extension.

What happens during the denaturation step of PCR?

During denaturation, heat is applied to the DNA template to separate the double-stranded DNA into two single strands, making the DNA accessible for further steps.

Why is the annealing step important in PCR?

The annealing step is important because it allows primers to bind (anneal) to their complementary sequences on the single-stranded DNA templates, which is necessary for the DNA polymerase to start synthesizing the new DNA strand.

How does the temperature affect the annealing step?

The temperature for annealing is crucial; it must be 3-5 degrees Celsius below the melting temperature (Tm) of the primers. If the temperature is too high, the primers won't bind efficiently, and if it's too low, there may be non-specific binding leading to unwanted PCR products.

What happens during the extension step of PCR?

During the extension step, the temperature is adjusted to the optimal level for DNA polymerase to add nucleotides to the 3' ends of the primers, forming the complementary DNA strand based on the template.

What factors determine the length of the extension step?

The length of the extension step depends on the extension rate of the DNA polymerase used and the length of the desired PCR product.

How many steps are involved in one PCR cycle?

One PCR cycle involves three steps: denaturation, annealing, and extension.

Why is it necessary to repeat PCR cycles multiple times?

PCR cycles are repeated multiple times to exponentially amplify the target DNA sequence, generating a large number of copies for further analysis or use.

What is the role of DNA polymerase in PCR?

DNA polymerase is responsible for adding nucleotides to the 3' ends of the primers during the extension step, synthesizing the complementary DNA strand based on the template.