Hi, I've some questions I can't find answers for and hopefully someone over her can point me in the right direction.
We have primersets against human targets, but our samples aren't clean (faeces/skin bacteria). Blasting these primers with NCBI using Refseq representative genomes for Homo Sampiens and Bacteria. This gives quit a list of Products on potentially unintended templates. I can't find databases about wich bacteria there are on human skin or in faeces. So I would like to know when are these product realy formed during PCR, on what minimum of nucleotids can a polymerase bind and start? 6bp on 3' end? If there is 1 mismatch will it still be possible to start? And what about the length of the unintended amplicon, many are >1000bp, I think there is not enough time in the extension phase the make such products, still it would influence the pcr efficiency right? due to primer binding/polymerase activity.
So basically which of the shown unintended products should I realy be worried about? And should find out it its in our samples or not.
Hi,
as you said the difference in Amplicon length is already a good point to start with. Assuming that your amplicon of interest is probably not longer than 500 bp and the unintended ones are above 1000bp you can use a shorter annealing extension time to favor the GOI amplification. Further, using the optimal annealing temp will help you to reduce unspecific or unwanted binding of your primers.
I would first try these two measures, if they are not effective you could think about adapting the primers using modifications like LNAs to increase the annealing temperature.
Good luck
Sven
Article Polymerase Chain Reaction: Basic Protocol Plus Troubleshooti...
Hi Timo
Sven is right. Annealing temperature and time are parameters you can set up to achieve a good specificity. Also magnesium concentration can be modified to get a better specificity, as well as the use of Hot Start Taq Polymerase instead of standard polymerase.
Did you try to blast your primer set in the Primerblast website?
https://www.ncbi.nlm.nih.gov/tools/primer-blast/
This is a good online tool to check for specificity, and it can give you some infos on melting temperatures of aspecific products (if present).
In any case, it is better to try your primer set by a PCR on a subset of your samples. Check on gels for aspecific bands, and even if only a band is present, try to sequence the amplicons to be sure it is human DNA. Good luck!
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