Polymerase Chain Reaction (PCR) is a widely used molecular biology technique that allows researchers to make multiple copies of a specific DNA sequence. There are two main types of PCR amplification: PCR thermal cycling and isothermal amplification. In this article, we will compare and contrast these two methods and discuss their advantages and disadvantages.
What is PCR thermal cycling?
PCR thermal cycling involves changing the temperature of the reaction mixture during the amplification process. The reaction mixture is usually heated to a high temperature (usually around 95°C) to denature the DNA into single strands. It is then cooled to a lower temperature (usually around 50°C-60°C) to allow the primers to anneal to the template DNA. The mixture is then heated again to a higher temperature (usually around 72°C) to allow the polymerase to extend the primers and create new complementary DNA strands. This process is repeated for 20-40 cycles to generate millions of copies of the target DNA sequence.
What is PCR Isothermal amplification？
Isothermal amplification, on the other hand, involves maintaining a constant temperature throughout the amplification process. The most commonly used isothermal amplification method is the loop-mediated isothermal amplification (LAMP) which is performed at 60-65°C. This method utilizes four primers and a strand-displacement DNA polymerase to produce a large number of copies of the target DNA.
Advantages of PCR thermal cycling
One of the main advantages of PCR thermal cycling is its high specificity, as the specific temperatures used in each step of the reaction help to reduce the risk of non-specific amplification. This is important when working with complex samples, as it helps to ensure that only the target DNA is amplified. Additionally, thermal cycling is highly sensitive, as it can detect even low amounts of target DNA.
Advantages of PCR isothermal amplification
On the other hand, isothermal amplification is much simpler and faster than thermal cycling. It requires fewer steps and equipment, making it a more cost-effective option. Isothermal amplification is also more suitable for field applications, as it does not require a thermal cycler and can be performed using a portable device.
Conclusion
Both PCR thermal cycling and isothermal amplification have their own unique advantages and disadvantages. PCR thermal cycling is highly specific and sensitive, while isothermal amplification is simple and cost-effective. The choice between these two methods will depend on the specific needs of the research project.
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