primer design - (Sep/26/2009 )

Hi,

i have a question about primer design.
I know it's necessary to check if your, designed primers, do not align towards hairpin structures, in your template. This can be done, using mfold.
About this concept, i have the following questions:
1) What if the hairpin structures have a positive free energy?
2) Is it a problem, if there are hairpin structures in the amplicon? With this, i mean, between the foward en reverse primer. I thought that maybe hairpin structures in the amplicon can interfere with the polymerase?

many thanks,

Waiting for answers..

greetz,

susan

Honestly, I never thought about any of this before designing primers. I use the program primer 3 (Link) to design primers and the algorithm seems to take care of all relevant stuff. Is it important for you to know this stuff or do you just need primers for RT-PCR? Just interested...

Stardust

susanna on Sep 26 2009, 04:33 AM said:

I think Positive Gibbs free energy means the formation of hairpin structure is unfavorable.

Hairpin structure in the amplicon (Secondary structure ) causes less product yield due to the polymerase being unable to complete extention . The extent of yield decrease depends on how stable the secondary structure is . However the extension temperature 72 is usually high enough to overcome this problem unless the secondary structure is too stable i.e. high GC content .

Secondary structure in Primers has more pronounced effects on yield since at the low annealing temperature Primers may "prefer'' to anneal to themselves rather than the target . However , I read a while ago that hairpin loops in primers could increase PCR specificity to some extent . That's because it would be energetically more favorable for Primers to anneal to themeselves rather than a mismatched region on the DNA strand .

Anyway , I suppose that a good Primer design software would automatically take care of all these issues and give you the best bet.

There is a very important difference between hairpins and dimers with 5' overhangs vs. ones with 3' overhangs. Since extension happens only at 3' ends, the 5' overhangs can act as templates for extending the primers. If this happens, the primers no longer match the template at the 3' end, and will fail to prime. This is the main problem with primer secondary or dimer structures. 3' overhangs don't matter as much or at all.

Yes, it may greatly affected your PCR

I have a pair of months trying to get the 2.5 kb product with a long hairpin (1.5 kb) in it.

phage434 on Sep 26 2009, 08:38 AM said:

I would highly agree with you . In fact another major difference between hairpins with 3' overhangs and hairpins with 5' overhangs is that in the latter the proportion of Primers available for annealing decreases constantly with each cycle because hairpins get further stabilized due to extension. while in the case of hairpins with 3' overhangs the proportion of Primers unavailable for annealing to target DNA remains constant in each cycle.

stardust on Sep 26 2009, 04:34 AM said:

hi,

actually i'm making primers for Q-PCR. But i want them to work well in theory, hoping to get better results in practice. That's why i'm checking them like that ;-)

susan