Good PCR and bad qPCR with the same primers

5 posts / 0 new
Last post
Johnny82
Johnny82's picture
Good PCR and bad qPCR with the same primers

 Hello everyone,
I ve been struggling with this problem for weeks!
I tested some primers with a temp gradient PCR and the results were very good from 59° up to 65°
timing 30, 30 , 40 seconds to better test  the specificity
but when I swithed to real time pcr (Roche) I found out many unspecific amplifications
even at high temp (65°)
These primers are designed to map a genomic region so I can't really change them much

Any idea?

Thanks!

Ivan Delgado
Ivan Delgado's picture
Hi Johnny,

Hi Johnny,

When you say that your PCR results were "very good" with your gradient PCR what exactly do you mean? If you ran your PCRs in an agarose gel and the results looked specific and then did the same and saw unspecific amplification using real time PCR, then it is very likely that what you are seeing is that real time PCR is many times more sensitive than standard PCR. In other words your PCRs were always non-specific, it is just that real time PCR is capable of detecting these non-specific bands while your standard PCR is not.

Having said this, if the non-specific amplification you are detecting by real time PCR is minor enough relative to the main amplicon you are interested in, you can still use this assay. Otherwise you may have to redesign your assay. I always suggest to test at least three different real time PCR assays to find one that works well.

Hope this helps. 

Johnny82
Johnny82's picture
 Thank you Ivan for your

 Thank you Ivan for your suggestion, I' ll try another brand of master mix then

besides I was wondering, is there a way to decrease the sensitivity?

Ivan Delgado
Ivan Delgado's picture
Hmm, decreasing sensitivity

Hmm, decreasing sensitivity is a somewhat odd thing to try since it kind of implies trying to make the assay less efficient. The only thing I can think of that would reliably decrease the sensitivity of an assay is to design multiple assays and chose the least sensitive one. 

Biju
Biju's picture
Johnny82

Johnny82
PCR primers may not be the best primers for Real-time PCR due to the increased sensitivity and also the differing set of parameters to select during primer design.If you cannot be flexible in pimer design, the best action could be calling technical support sepcialists at Roche or Invitrogen and get their help.
Thanks
Biju Joseph