for example we have a DNA from E. coli and we have a specific primer pair for X gene. when we perform the PCR and check the PCR products we found a non specific products in addition to the product of interest X gene. what is the best explanation?
Non specific annealing of the primers. Especially if the annealing temp is low. In such case, they can stick to a non target part of a genome.
Try temp gradient to find the optimal annealing temp.
Non specific annealing of the primers. Especially if the annealing temp is low. In such case, they can stick to a non target part of a genome.
Try temp gradient to find the optimal annealing temp.
Yes, Tannealing optimization is the first step you should do, but quite often, the problem cannot be fixed by higher Ta - when the primers were designed for target gene of model Ec strain without searching for homologous sites across many Ec genomes (true for some old works).
if you have a pseudogene or similar gene family member or even contamination of the dna from another species then this can be a problem. For non specific priming you can try adding DMSO or temperature gradient pcr or touchdown pcr . Sometimes one primer has analogy with a repeat sequence and just one primer can be close to itself in reverse orientation and a repeat product is produced. In this case pcr works with just one primer...try it and if you get a product with only one primer then re design that primer
You can try a touchdown pcr starting from high annealing temperature like 63-65 and gradually decreases with each cycle.
And also try hot start PCR.
Following onto Paul Rutland's reply, it's always a good exercise to BLAST your primers and look for any potential red flags that in silico algorithms might detect.
optimize temperature condition, go for BLAST to find any homologous sequence
Primer dimers are your problem. Try the optimize your annealing temperature and optionally you can try touchdown pcr.
In case your specific primers have a short sequence then the chances of non specific binding is higher. Gradient PCR might help you in finding the optimal conditions.
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