what is the temperature used for the extension step?

2 min read 26-09-2024

what is the temperature used for the extension step?

Polymerase Chain Reaction (PCR) is a widely-used technique in molecular biology to amplify DNA. One of the critical phases of PCR is the extension step, where a specific temperature plays a vital role in the efficiency and effectiveness of the process. But what exactly is the temperature used for the extension step, and why is it so important?

What is the Extension Step in PCR?

The extension step occurs after the denaturation and annealing phases of PCR. During this phase, a DNA polymerase enzyme synthesizes new DNA strands complementary to the target sequence. The most commonly used polymerase in PCR is Taq polymerase, derived from the thermophilic bacterium Thermus aquaticus.

Key Points of the Extension Step:

Temperature: The extension step typically occurs at 72°C (162°F).
Purpose: This temperature allows the DNA polymerase to effectively add nucleotides to the growing DNA strand.

Why 72°C?

The choice of 72°C for the extension step is based on several factors:

Optimal Activity of DNA Polymerase: Taq polymerase, and many other DNA polymerases used in PCR, exhibit optimal activity at temperatures around 70°C to 75°C. At this temperature, the enzyme can efficiently catalyze the polymerization of nucleotides.
DNA Stability: While DNA strands are denatured at higher temperatures, 72°C provides a stable environment for the double-stranded DNA to remain intact while allowing the enzyme to function effectively.
Extension Time: The length of time for the extension step can vary depending on the size of the target DNA. Generally, it's advisable to extend for 1 minute per kilobase (kb) of DNA. For example, if you are amplifying a 1 kb fragment, you would typically extend for about 1 minute.

Variations in Temperature

While 72°C is standard, some protocols might use slightly different temperatures based on the specific DNA polymerase or the application. For instance:

High-Fidelity Polymerases: These may function optimally at slightly lower temperatures (e.g., 68°C) to enhance the accuracy of DNA synthesis.
Long-Range PCR: When amplifying longer fragments of DNA, some protocols might employ higher temperatures (up to 80°C) to facilitate the activity of specialized polymerases that tolerate these conditions.

Practical Example

Imagine you are performing PCR to amplify a segment of DNA that is 2 kb long. Following the denaturation (94-98°C) and annealing (typically 50-65°C) phases, you set your thermal cycler to 72°C for 2 minutes for the extension step. This ensures that the Taq polymerase has adequate time to synthesize the entire 2 kb fragment efficiently.

Conclusion

The extension step temperature in PCR is a crucial component that significantly impacts the amplification process. Using 72°C allows for optimal enzyme activity while ensuring the stability of the DNA. As you develop or optimize PCR protocols, considering the specific needs of your experiment, such as the length of the target DNA and the type of polymerase used, is essential for success.

Additional Insights

To maximize your PCR results, consider:

Template Quality: Ensure that your DNA template is of high quality and free from contaminants that could inhibit polymerase activity.
Buffer and Additives: Using the correct buffer and additives like glycerol or betaine can enhance PCR efficiency.
Cycle Number: Adjusting the number of cycles (typically between 25-35 cycles) can also impact yield and specificity.

By understanding these parameters, you can improve your PCR results and achieve more reliable amplification of your DNA targets.

References

This article is based on information derived from questions and answers found on ScienceDirect, attributed to their original authors. For more detailed and specific guidance on PCR techniques, consider referring to their publications or protocols.