Every PCR reaction has the same problem:
Very little amplification of target DNA, but more amplification of non-target DNA. However, you can solve the problem of your PCR reactions by using some additional reagents. It is up to you to determine which PCR reagent does what and which condition is best for you.
PCR extension reagents generally work in one of the following two ways:
① By reducing secondary structures and increasing amplification of target DNA ↑
② By reducing non-specific binding and decreasing amplification of off-target DNAs ↓
Mg⁺² is required for polymerase activity. Withoutsufficient Mg⁺², taq polymeraseremainsinactive.The correctconcentration of Mg⁺² increasesspecificity. LowMg⁺² concentration increasesnon-specific binding.
Bovine serum albumin is a commonly used addition in various molecular biology applications, especially in restriction enzyme digests and PCR. In PCR, BSA can help combat contaminants such as phenolic compounds. It is also reported to prevent reaction components from sticking to tube walls.
Improves amplification of GC-rich and difficult DNA targets. Ideal for use on sequences known to be difficult to amplify. Reduces DNA Tm temperature.
The use of chemical components such as Triton X-100, Tween 20, NP-40 is also thought to reduce secondary structures. The use of these reagents may increase PCR efficiency but may also increase non-specific amplifications. Therefore, they should be used with caution.
It is thought to reduce secondary DNA structures. Therefore, it may be advisable to add GC-rich constructs during PCR analysis. However, DMSO can also cause a decrease in Taq polymerase activity, so it is very important to balance the two.
It is an organic PCR extension reagent. It works to clean up dirty PCR reactions by reducing non-specific binding.
Although all these components improve PCR results, it is not possible to predict at the first stage what is best for your analysis. It takes a lot of experimentation to optimize a PCR reaction.