Hello everyone
I need your help with a problem I can't seem to solven :
I'm planning to do some sequencing of freshwater algae. So I referred to the primer pair made by Stoeck et al. 2010 and Balzano et al. 2015, which is supposed to be general, according to several articles I've read, and quite effective:
Forward primer: V4F (5'-CCA GCA SCY GCG GTA ATT CC-3')
Reverse primer: V4RB (5'-ACT TTC GTT CTT GAT YRR-3')
However, after testing several different PCR cycles and checking on an agarose gel, I very rarely obtain a single band of ~400bp (the desired size).
Most of the time, I end up with either no migration band or several other non-specific bands, including one that is 300bp larger than the desired band.
You can check that on the picture.
I have used the cycles recommended by several articles using these primers (Salmaso et al 2020, Latz et al 2022, Balzano et al 2015...), but I don't get any satisfactory results.
I also carried out several tests with different hybridisation temperatures, reduced the proportion of DNA in the PCR mix, added DMSO and reduced the number of cycles, but these did not give satisfactory results.
But unlike most of the articles that use KAPA HiFi HotStart, the basic polymerase in the Swedish studies, I use pHusion HF HotStart Polymerase.
- Do you think these non-specific amplifications could be linked to the difference in polymerase?
- Have you ever had this kind of problem with primers?
- What do you recommend?
Thank you very much for any help you can give me.
Good luck with your research !
Thomas Charpentier
Check the temperature The average Tm of both primers should be used as the annealing temperature in PCR. Increasing the annealing temperature can reduce non-specific reactions and The DNA sample may not be pure enough, so it may not amplify in certain strains. You can try washing and re-precipitating the sample DNA to remove contamination. Identifying microalgae, you can use 18S rDNA for eukaryotic microalgae and 16S rDNA for prokaryotic microalgae.
You can cut the bands, extract them and re-amplify them separately. Each band can be sent for sequencing and you can blast to find out if they correspond to the same region. sometimes there are very large differences between alleles due to very large indels.
If they are different regions you can use the same technique and sequence only the 400bp band.
It occurs to me that it could also be a case of contamination by DNA from another organism (probably fungi) in the sample, in the primers or in another pcr product.
Hmm, it could be a primer issue. I found an alternative reverse primer with a bit less degeneracy at the 3' end. Here is the article and their methods section.
Green microalgae in marine coastal waters: The Ocean Sampling Day (OSD) dataset | Scientific Reports (nature.com)
V4 was amplified using TAReuk454FWD1 (5′-CCA GCA SCY GCG GTA ATT CC-3′) as forward primer and the modified TAReukREV3_modified (5′-ACT TTC GTT CTT GAT YRA TGA-3′)
Could be a polymerase quirk too, I've found Fusion to be a bit finicky. Mostly I use Q5 from New England Biolabs, have had good success in the past with Vent.
I also agree with Mauro Sanna about sequencing the bands to find out what you are amplifying.
I agree with Amy, shortening the extension time could help. I use the same V4F as you, slightly shorter V4R (ACTTTCGTTCTTGAT), Q5 polymerase (NEB) with 30s extension and I see no non-specificities. So it could be a matter of polymerase / buffer.
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