We are working with DNA samples extracted from faeces, so we are trying to find the optimal PCR conditions to amplify microsatellite markers, as the nature of the samples require quite an effort.
On the image with the stuttering zones is represented the second test we performed in order to see what conditions are optimal.
We used the same PCR conditions with the expection of the following:
-Finan Extension was a)68 oC b)72 oC for both for the duration of 30min as stated by the Qiagen PCR Multiplex Kit protocol
-T annealing both times was 60 oC
What could cause such an utter mess? Is it possible the higher temperature on the final extension step do this?
Is the y scale the same in both images? To me it looks like the "mess" might be present at the lower extension temperature as well, just at lower intensity relative to the main peak. I can't really even see your standard peaks on that first trace.
I've looked at a lot of microsatellite traces for a lot of different species and can honestly say I've never seen that before. My best guess is that your primers are annealing to multiple spots in a repetitive region. My advice would be to drop that marker from your panel and move on. It will be hard to optimize with that as your start point.
Good luck and post back if you solve the riddle!
Joel D Anderson yeah.. i was trying to correct the mess at the lower extension temperature.. I added fewer DNA volume and perfofmed fewer cycles on the second trial due to the fact that the standard peaks are too low.
It might be the case you suggest but it is striking the differnce between the two trials.
Will do, and thank you for your reply!
Extension temperatures may not be the most relevant here.
You should first try that pair of primers in a “clean” sample with only DNA from the target species. Which species are you targeting? What is the ideal annealing temperature?
Then, perhaps using another marker or some DNA quantification method, determine an ideal DNA concentration for your type of sample for the PCR. You may have an excess of DNA.
Then, perform the PCR with the highest possible annealing temperature, perhaps a gradient could help to determine that ideal temperature.
Don’t forget, dinucleotide repeats are prone to that stutter pattern. The multiple peaks may be also some unspecific amplification, likely to occur in mixtures and at high DNA concentrations and suboptimal annealing temperatures.
Hi Filipe,
Thanks for your suggestions. The problem exists on hair samples of the species,too. On the first trial primers seem to work just fine, not only on hair ("clean") but also on stool extracted DNA samples. It's an ungulate species R.r.balcanica.
As fas as the annealing temperature goes, the optimal is 59oC.
One thought is that the cycles may play a role for this mess. On the first trial I performed 45 cycles and on the second 40 cycles. It might be possible due to the mixture that our target DNA was not amplified enough..
Best,
Nikos
The anneling tempteature is the important is important try diffrent temreature
The anneling tempteature is the important is important try diffrent temreature
The tempreature is the important is important try diffrent temreature
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