How can I avoid primer-dimer formation during PCR amplification?
Posted January 18, 2023
There are several things that can be done to avoid primer-dimer formation during PCR amplification. One thing that can be done is to avoid primer-dimer formation by creating optimal primer pairs and using proper primer concentrations. Using a sequence of additional nucleotides at 5’ ends of amplifiers is one example. Tailed amplifiers occur at low concentration and are only involved in the early steps of PCR. Primer-dimers have a high concentration of complementary sequences from the tail and thus causes the annealing of the complementary ends of a single strand by tailed primers. This forms pan-handle structures and this subdues the annealing of Tag primers and prevents primer-dimers from forming. Additionally, during PCR amplification complementary sequences of two or three bases at 3’ ends of primer pairs should be avoided; complementary sequences between a primary sequence and primer pair should be avoided as well. The primer concentration should also be reduced to the minimum at which amplification can occur. Additional steps can also be done such as adjusting the annealing temperature and using a PCR enhancer like BSA or DMSO. It is also important to check the template as high concentration and low purity can cause dimer formation.