Hello, I am trying to characterize some microalgae strains and I am doing PCR using primers for the ITS-2 region. In some strains I obtained 2 PCR products: one is in the expected range of size (600-1000 bp), the other one is higher and I really don't know what it is.
Anyone has experience with the amplification of this region?
Hi
I think you have two primers fpr 18s rDNA characterization- ITS-1 as forward and ITS-2 as reverse. When you amplified in PCR you have to take care about Tm of both primers. The average Tm of both primes should be kept as temp for annealing step in PCR so that you can get more precise results.
other possibilities, Sample DNA may not have enough purity and hence it may remain unamplified in certain strain where you found higher bp bands. This can be rectified by washing and re-precipitation of sample DNA to remove contaminations (Protein, Phenols etc...!!!) followed by PCR amplifications.
I know that my DNA is not pure because I am doing colony PCR. I am following a paper that shows how colony PCR is a rapid method for taxonomic studies through amplification of this molecular marker (ITS-2). And I am obtaining good results. The only problem is this second band, higher that 1 kb, that I have encountered for same strains.
To my experience, colony PCR may be useful for bacteria (DNA may be extracted by heating the bacterial colony at 95C for 5 min.). But, microalgae has a different cell composition so you can try to isolate DNA from algae by Fungal DNA isolation kit which available ready to use in market.
There are some papers showing colony PCR is also suitable for microalgae... For DNA extraction I used a good and rapid protocol but colony is the fastest way for PCR screening...
Of course:
Liu et al. Single-tube colony PCR for DNA amplification and transformant screening of oleaginous microalgae. J Appl Phycol (2014) 26:1719–1726
Packeiser et al. An Extremely Simple and Effective Colony PCR Procedure for Bacteria, Yeasts, and Microalgae. Appl Biochem Biotechnol (2013) 169:695–700
Radha et al. Direct colony PCR for rapid identification of varied microalgae from freshwater environment. J Appl Phycol (2013) 25:609–613
Wan et al. An improved colony PCR procedure for genetic screening of Chlorella and related microalgae. Biotechnol Lett (2011) 33:1615–1619
The first paper is the one I following most, but I changed some steps.
Well, I suppose that you are using some primers for ITS region, not specifically for ITS2. After that, for example, when I did the PCR for ITS region in Tetraselmis, also I found two different bands. The first one is for ITS, the second is some unspecified band, that also is very common when you try with polysaccharids-producer microalgae. If you are trying to isolate the ITS region for taxonomic classification, try to obtain those two bands, and then isolate the ITS band by cutting the electrophoresis agarose gel for purifying. Take account that usually some microalgae (e.g. Tetraselmis marina or some red m icroalgae like Porphyridium) has a lot of polysaccharids that interfer with the PCR reaction. So, if is this the case, before the DNA extraction you can trying extracting the polysaccharids with isopropanol or some DNA columns for purifing the sample. In your colonies, do you have different strains of different "species" of microalgae? just one?... Cheers!
Can you purify that larger band and sequence it? Then you will know what‘s that.
The non-specific amplification happens very often in ITS-PCR. Usually the template amount of colony PCR is hard to quantify and sometimes exceeds the optimistic concentration, which will probably lead to the non-specific amplification.
In your case, as long as you are getting the results you want, you don't really have to optimize the PCR conditions.
Hi Lorenza Ferro
May i know, what is the sequence for the primer that you had been used? I really hope you could tell me.
Thank you.
Hi Nurul,
please, refer to this paper https://www.researchgate.net/profile/Jin_Liu45/publication/259539047_Single-tube_colony_PCR_for_DNA_amplification_and_transformant_screening_of_oleaginous_microalgae/links/53da4be50cf2631430c8108f.pdf
you will find the primers sequences I used.
Good luck.
Lorenza
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