Hi Binbin

Usually the denaturation temperature of 94C should eliminate most of secondary structures of DNA. I would say a 15bp stem could not stand that temperature.

Hi Binbin,

Actually, secondary structures affect to PCR.

It decrease primer binding on template by binding themselves(hairpin).

But in some case, they prefer to bind to your template than themself.

So, you can get target product but it will be less than your expection usually.

Althogh I recommend to use primer have no secondary structure, also I think you can try.

Cheers.

run your pcr in presence of 1Molar betaine or any amount od DMSO up to 8%. Either or both of these will minimise secondary structure and I would expect the pcr to work. Also having long primers annealing at a high temperature will not allow secondary structure problems to interfere too much

Hi Ali Javadmanesh ，

Thank you for the answer!

I check the thermal stability of the hairpin on IDT online oligo analyze, the melting temperature of the hairpin structure is more than 70C. Does it mean the template may prefer forming intramolecular than forming hybrids with the primer pairs and TaqMan probe?

Will the self-folding of the template during the annealing process occur before the primers bind to the sequence since the melting temperature of he hairpin structure is much higher than that of the primer binding.

Thanks

Binbin

Hi Binbin

Now it sounds like a problem. What is the annealing temperature of your primers? Most of times I use more than 25n in length to design a primer thus my Ta would be around 60-62. Although you may have a bigger problem probably will be solved by PCR enhancers such as betaine & DMSO as Paul Rutland recommended. To be honest I never cared about template secondary structures after designing 100s of primer pairs.

Goodluck

Hi Paul Rutland and Ali Javadmanesh ,

Actually I already add 1M betaine to my reaction. In the beginning, my TaqMan assay suffered from a upward baseline shift. After addition of the Betaine, the problem is well addressed. But somehow, the PCR efficiency is not good all the time. I have been trying to optimize the protocol, but there seems to be little effect. Since my template has very complex secondary structure, I just suspect the secondary structure has some influence in the PCR.

Do you know any papers or books that explain the effect of the secondary structure of the template on the PCR?

Thank you very much for the help!

Binbin