An unexpected but common problem in PCR is primer melting, which is an unwanted result of the repetitive heating and cooling. Primer melting could also lead to a decrease in product yield as well as potential false sequences. So how do you prevent it? If you know what type of primers you will be using and what kind of sequence, then make sure they are compatible with each other for high specificity and accuracy.
In short, the melting temperature is the temperature at which a sequence will begin to dissociate from its complementary strand. It is apparent that this melting point plays an important role in primer selection and designing, especially in PCR. The melting point depends on several factors: primer length, base composition and sequence of the base, as well as the conditions of the reaction such as MgCl2 concentration. Oligonucleotides that are 17 to 25 nucleotides away from each other should be separated with at least 2 nucleotides from all other primers. It’s also important to note that primers can only be used a maximum number of times. This is especially important when the difference between the melting points of your primers and your target sequence is very small.
So what are you supposed to do if your primer design goes wrong? One way is to buy new primers from somewhere else, but that could be expensive. Another way is to optimize and alter the melting point of your primers so that you can prevent primer melting if it does occur in an undesired manner. There are several ways to optimize and increase the melting temperature of your primers.
One popular way is to use MgCl2 solution. The melting temperature of different oligonucleotides with the same base composition can be increased by adding MgCl2. However, it is important to note that MgCl2 solution does not have a very high concentration but instead 2-3%, which is not sufficient to increase the melting point of primers.
Another way that I am aware of, which consists in glyceraldehyde and formaldehyde is another way to increase the melting point of your primers by 50% or more. It takes about an hour for this process to complete, which is inconvenient if you have many reactions to complete in a short period of time.
Another popular way to increase the melting temperature in microliters is by adding BSA solution. BSA is an abbreviation for Bovine Serum Albumin, which is a protein that contains several essential amino acids and also calcium. It looks like this:
BSA is understandable as a possible source of calcium because it can be used to bind calcium ions and increase the heat stability. However, BSA cannot be used if your primers do not have high GC content. This can happen if you use wild type M13 DNA as your host rather than M13 mutagenesis vector or others with better GC ranges. The melting temperature of primers will increase about 25% in the presence of BSA.
In conclusion, it is important to understand the primers you are using and the sequence. Take special note of the melting temperature of your primers and ensure that they have a higher melting temperature than your target sequence. BSA and MgCl2 solutions are popular ways to increase the melting temperature of primers, although it is important to not use MgCl2 solution if you have too many reactions to complete in a short period of time. Glyceraldehyde and formaldehyde solution is another way, which I think works faster than BSA because it only takes about an hour for the process to complete.
Make sure your primer design is carefully planned before PCR! An unexpected but common problem in PCR is primer melting, which is an unwanted result of the repetitive heating and cooling. Primer melting could also lead to a decrease in product yield as well as potential false sequences. So how do you prevent it? If you know what type of primers you will be using and what kind of sequence, then make sure they are compatible with each other for high specificity and accuracy.