I adopt primer designs from a study published in Nature ( Article Domestication selected for deceleration of the circadian clo...
), so the primers should be qualified. Yet in many samples, I got nonspecific amplifications, with a second peak or sometimes completely shifted peak in the melting curve (picture). The strange peaks were consistent among 3 replicate runs of those samples as well. Some samples were fine, so I don't think temperature setup is the problem either.So what can be the cause of this?
Picture: Melting curves of 3 different samples (after qPCR of TOC1 gene). Lines with same color represent 3 replicates of the same sample. D1-3: supposely fine sample; D4-6: sample with a second nonspecific peak; D7-9: sample with completely shifted peak.
Depending on your source material, there is a possibility that this peak shift could be caused by a significant genetic variation. If such a picture is stable across series of samples, it makes sense to check some of them by sequencing the amplicon.
On the other hand, primers still can cause non-specific amplification with your particular equipment. It is good practice to perform PCR optimization even if primers are originated from a trusted source, for example, gradient PCR run in some range outside initial temperature.
Depending on your source material, there is a possibility that this peak shift could be caused by a significant genetic variation. If such a picture is stable across series of samples, it makes sense to check some of them by sequencing the amplicon.
On the other hand, primers still can cause non-specific amplification with your particular equipment. It is good practice to perform PCR optimization even if primers are originated from a trusted source, for example, gradient PCR run in some range outside initial temperature.
Could be a sequence variation inside the amplified region.
There are two slightly different nucleotide sequences given for TOC1/APRR1:
https://www.arabidopsis.org/servlets/TairObject?type=sequence&id=428247
https://www.arabidopsis.org/servlets/TairObject?type=sequence&id=428369
You could cross-check where your primers align.
Sequencing the PCR product might help to find out what's going on here.
Alexandr Pozharskiy
Jochen Wilhelm
I did do some test runs with the primers, and during the tests the amplifications were completely fine, with one single consistent peak for each gene. In fact, the majority of the samples were fine. Imagine my bewilderment when I checked my data and found out there were some samples with this peak shift/second peak, while the protocol has never changed.
I checked the data again and it does seem that the melting temp. of these shifted peaks are consistent across samples. Interestingly, in samples with 2 peaks, the second peaks also match these shifted peaks (you can also see that in the picture). This suggests that the products of these runs were not exactly nonspecific, and there are 2 variations of the mRNA and some samples contained both variations. Could this be the result of RNA splicing? Or is there any other possible explanation?
Duy Minh Pham
This can be allelic variations in the sequence. In that case double peaks correspond to heterozygous genotype
No, I wouldn't think that RNA splicing is a reason. RNA editing might mire likely a possible reason. But in the end, this results in sequence variations, and you when cooling down to the last cycle or the start of the melting corve, you get a mixture of homo- and heteroduplices that have slightly diffent melting temperatures (same if you have a genetic polymorphism).
I think what you are seeing is genetic variation in the TOC1 gene in your different tomato strains - SNP, small InDel, etc.
Sequence your PCR products and you'll know for sure.
If yes, then you have discovered a potentially useful genetic marker.
Even on taking primers from literature - did you crosscheck them by BLASTING for specifity by using Primer-Blast? Trusting is good, cross-confirming things is always better and a critical mindset is never wrong. I have seen in more than one publication that primer were completely wrong, they targeted a completely different gene that they were stated. And as reviewer of journals, I also critized more than once wrong primer sequences, but a lot of people miss it.
Next, did you load the PCR product on your gel to verify the size of your product? To check if the band size might be different in between the different PCR products?
Sequencing the PCR product would be the next step to get an explanation for the difference in the melting curve.
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