I am recently using a primer set to amplify some DNA 16S rRNA from soil. One primer has one base pair mismatch at the 3' end. I would like to know how will that one base mis-match at the 3' end can compromise DNA polymerase reaction. Will it inhibit amplification completely or partially?
Depending on the anealing temperature you can will have complete or partial inhibition.
Under optimal conditions for the specific primer you can expect a difference of up to 12 Cqs for a single 3'-mismatch. It depends on nature of mismatch and sequence context. Also if you use a 3'-5' exonuclease proficient enzyme, you will get a smaller delta Cq.
Cqs is a term taken from "real-time quantitative PCR" field. It essentially means that only each 2^(12-2) = 250th target molecules will effectively be amplified. If you want to use a 3' mismatch for allele-specific PCR you could use a 3'-C-alkylated primer that considerably increases the specificity.
http://www.ncbi.nlm.nih.gov/pubmed/14669997
One thing is overlooked here. We all know about Tm and annealing temperature, yet another factor plays here. DNA is elongated from 5' tot 3'. As a consequence a 3' mismatch can hamper DNA synthesis much more then a 5' mismatch. As in this case its only one, I would just try it.
Thanks everyone, I used https://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/ to estimate the annealing temperature, which is about 63 degree C. I then try a temperature gradient PCR There is no amplification at 63 C annealing temperature, but there is amplification at 60 C. Since I am working with environmental DNA from soil samples, I don't know what the PCR has just amplified. The sensible thing to do next may be some cloning and sequencing.
I would advise you to make a gradient PCR to check the best annealing temperature. Normally, if you decrease a bit the annealing temperature, you promote the non specific annealing. With a gradient PCR, you check which temperature is the best compromise between specific and non specific annealing. Start from 56°C until 63 °C and check on agarose gel if you find your PCR product or non specific amplifications, then choose the best annealing temperature.
in this case your pcr product depend many factors such as mgcl2 concentration, annealing temperature, and sort of mismatches. for example if have g/t mismatch it doesnot matter and hada little effect on your amplification .
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3174695/
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