One of the most common problems we face in #PCR is the formation of primer dimers, generally seen below the 50bp marker. These pose problem not only for conventional PCRs, but qPCRs as well as in #sequencing reactions. These are stable duplex DNA structures that amplifies with the help of #Taq DNA polymerase. These are of two types: cross (where there is #complementarity between forward and reverse primers) and self (where there is complementarity in the same #oligonucleotide, such that it loops and forms a duplex DNA structure).
FIGURE: It shows the appearance of #primerdimers in a template-gradient PCR. Note how decrease in template DNA amount increases the appearance of primer dimers.
What went wrong?
a. Use of excess primers.
b. Primers having G/C in the 3' region.
c. Self-complementarity in primers.
d. #Taq polymerase just needs a free 3' end and Mg2+ ions to start adding dNTPs even at lower temperatures, which causes primer dimers. It can cause both self and cross variants.
e. PCR additives/ enhancers such as excess MgCl2, #formamide, KCl etc. can also contribute to the issue.
f. The more the PCR cycles (beyond 35), the more the chances of getting primer dimers!
g. Too low of annealing temperature is also a cause.
Solutions?
1. Set up the reaction on ice, this will not let the Taq keep adding dNTPs to primer secondary structures
2. Use a good primer design software. As G and C are strong matches, avoid having G/C at the 3' end of the primers. #Blockcleavableprimers or #chimericprimers have also found use in this regard.
3. Wise use of PCR enhancers like excess MgCl2 which usually enhances amplification.
4. #HotStartPolymerase can be a stable solution. Here the Taq is bound to antibody or wax beads, and on reaching the ~98C temperature the bond breaks and the Taq can start doing work on the denatured, primer-bound template DNA.
Hope it helps!
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