Would the PCR reaction be hindered if my primers can form secondary structures at a temperature that is close to the primers' melting temperature? Since the temperature difference is small, the secondary structures could probably form even before the annealing temperature is reached (assuming the Ta is 5°C below the Tm).
The possibility is high that the yield of the PCR is low or that you will get primer dimers or similar products. We are working since several years with apoE, a GC rich gene, and we often had problems with primers with a high prediction of secondary structures. We tried to get rid of the additional bands by using DMSO, Q-solution or other additives and the lowest annealing temperature was 68°C.
1. Presence of the primer secondary structures produced by intermolecular or intramolecular interactions can lead to poor or no yield of the product. They adversely affect primer template annealing and thus the amplification. They greatly reduce the availability of primers to the reaction. http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html
2. Add DMSO or Betaine or both to your PCR reaction as mentioned by Michael, they improves PCR amplification specificity of DNA with complex secondary structure. I use DMSO.
@Olga
Tm = Melting temperature
I didn't calculate the Tm of the secondary structures. Rather, it was an extra bit of info that came with the parcel containing the oligos. dG is -1.46.
@Yuan-Yeu Yau
I was asking what is Tm for primers, not the definition of the Tm. Thank you though!
dG=-1.46 is ok. Standard PCR should work just fine!
Can you show the pairing?
If you analyze your primer sequences with Primer 3 at http://bioinfo.ut.ee/primer3-0.4.0/primer3/ , you will get info on the stability/matches of the 3' ends of the primers with themselves and with each other. It's very unlikely that the melt point of a primer secondary structure would be anywhere near the melt point of the primer paired with its complement, so the more typical concern is whether primers will "prime" themselves or each other at their 3' termini. This could lead to extension by the PCR polymerase and the occurrence of a "primer-dimer" product that will compete with the desired product amplification.
"I didn't calculate the Tm of the secondary structures. Rather, it was an extra bit of info that came with the parcel containing the oligos." - Very interesting. Which company sends such info ?
Sometime strong secondary structure will hinder PCR amplification. In that case, adding DMSO to reaction mix will help.
Charith Raj Adkar-Purushothama , how much concentration of DMSO can use for proper PCR amplification if we found strong secondary structure?
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