SYBR Real time PCR - peak with shoulder - what to do? (Dec/05/2007 )
hallo all,
have a problem with melting curve. am i allowed to use results of a SYBR real time PCR when the melting curve has a shoulder?
i´m a little bit in a pressure of time (preparation of a presentation) and don´t know if it is time to repeat all my PCR´s for that gene (playing with annealing times and primer conc).
what should i do?
A shoulder on a melting curve usually show primer dimers. You can try to put less primer and hope that the shoulder disappears.
It could also be a non-specific product. Have you checked the PCR products on gel?
hey
I agree; to trust your data, the shoulder needs to be gone. that will decrease your Ct for that product, skewing all your results. I'm sorry!
have a problem with melting curve. am i allowed to use results of a SYBR real time PCR when the melting curve has a shoulder?
i´m a little bit in a pressure of time (preparation of a presentation) and don´t know if it is time to repeat all my PCR´s for that gene (playing with annealing times and primer conc).
what should i do?
What Tm is your shoulder? What qPCR machine? (lightcycler?)
If you run a gel and confirm product specificity, and your shoulder is at a Tm indicative of primer dimers (ie: about 80C) (ideally you also sequence your target or otherwise validate but alot of people dont do this) Then I think you can use a trick to make this usable. The thing important for calculating efficiency of amplification during the reaction is the concentration of double stranded DNA containing sybrgreen at the time of acquisition (the end of each cycle) when it is read. By heating the reaction to a temperature above the Tm of your primer dimers but below the Tm of the product (81C is typically used) then at the time of data acquisition it will be as if there are no primer dimers at all. If your dimers are severe, indicating that there is enough primer interaction to actually negatively impact the amplification of the specific target then you will only be okay if you are using a lightcycler which can correct for efficiency in the reaction (and still if your efficiency with dimers is like 1.9 and it goes to 1.6 with the 81C acquisition this means you have a primer problem and you have to re-design.) I think it is worth a trial run to just change the acquisition temperature and see if it will work...
Anyway just a suggestion, may not help with time, but you may be able to present the data you have as qualitative not quantitative in the meeting and then re-run after?... Again totally dependent on your having a single specific product and the shoulder is really primer dimers...
hope this helps and good luck!
Ok, there's one other possibility. I know it's not probable but it has happened to me. If you are amplifying cDNA and your amplicon has a SNP, it might show as a peak with a shoulder. In these cases, when checking the PCR product in a gel you will see just one band. You can check by sequencing your PCR product.
How can SNP produce a shoulder on your curve?
One copy of a cDNA should not be enough to produce a shoulder over a population of molecules, unless the SNP occured soon on the PCR.. And even then..
If you have a SNP in your sequence and your sample is heterozygous for that SNP, then in theory you will have 50% cDNA target molecules from each "genotype", so yes, if the SNP produces a substantial change in the Tm you will see a shoulder or even two distinct peaks in the melting curve. Actually, this is the basis for allelic discrimination using real-time PCR...
DNA with different GC content will have substantially different Tm. If you have a region (30 - 50 bp is enough) of your PCR product with dramatically higher or lower GC content than the remainder, it is easy to get a shoulder in the melting curve, even with perfect amplification and no primer-dimers. If you have a single band in the pcr product, then the shoulder is not important.
I checked pcr product on agarose gel. there are two bands (1 real big band, and 1 faint band 100bp below) at 2 highest standards (100ng, 10ng). below only one band is available, but maybe due to limited sensitivity of detection.
Then I think that your PCR product specificity is compromised (ie: your shoulder is not primer dimers but rather a non-specific or misprimed amplimer) This means that you will have to change your PCR conditions to get a single product of the expected Tm/size. Usually increasing annealing temperature, changing Mg++ concentration or addatives like DMSO are used to get better specificity.
Hope this helps and Good Luck!