I have used IDT Oligoanalyzer for checking my primers. In here, the accepted delta G value for internal self‐dimers(-9kcal/mol) and the 3' end (-6kcal/mol) has been mentioned but not for the 5'end dimers. My primer, after addition of restriction enzyme site, shows dimer at the 5' end with a Delta G of -9.96 kcal/mol. With addition of any R.E. site, the same trend is being seen. Is it going to affect my PCR? Does 5'end dimers affect PCR?
Hi Tahbildar,
The key parameter of ΔG usually focus on 3' end of the primer dimer which can extend to generate non-specific amplification. Besides the amplifiable cross dimer on 3' end, the cross dimer on 5' end may cause to poor product. Because the excessive primers can anneal to each other and have impact on annealing on target region. I don't find the paper for this part, but there is one website that mentions the requirement.
Cross Dimer: Primer cross dimers are formed by intermolecular interaction between sense and antisense primers, where they are homologous. Optimally a 3' end cross dimer with a ΔG of -5 kcal/mol and an internal cross dimer with a ΔG of -6 kcal/mol is tolerated generally.
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