I am working on some plants from Valerianaceae family(angiosperms). I have already sequenced rbcl, matK and two inter-genic spacer regions. I want to amplify ITS1 and ITS 2 regions. Here, previously, I have used primer originally developed by White et al. They didn't work because after sequencing I use to get fungal and bacterial sequence in BLAST hit. So, I am now using ITS2-S2F developed by Chen et al. and ITS 4 primer to amplify ITS 2 region (referred by CBOL working group). Now, I am not getting fungal/bacterial sequences, but getting sequences from Chlorophyta (like Chlorella etc). Every time I used negative control there was no bands present.
If there is any contamination in my samples how can I get all the sequences and nice BLAST hit?
I guess you could design your own primers. Download from Genbank what you can find of ITS1/5.8S/ITS2 sequences for Valerianaceae/Caprifoliaceae, and align them. Then look for conserved regions suitable for primer design. I would design (a couple of) forward primers at the beginning of ITS1 and at the beginning of 5.8S, and a (couple of) reverse primers at the end of ITS2 and at the end of 5.8S.
You can then test PCRs using different primer combinations, including also the already published primers that you have tried. Hopefully this will allow you to obtain at least partial sequences for ITS.
I guess you could design your own primers. Download from Genbank what you can find of ITS1/5.8S/ITS2 sequences for Valerianaceae/Caprifoliaceae, and align them. Then look for conserved regions suitable for primer design. I would design (a couple of) forward primers at the beginning of ITS1 and at the beginning of 5.8S, and a (couple of) reverse primers at the end of ITS2 and at the end of 5.8S.
You can then test PCRs using different primer combinations, including also the already published primers that you have tried. Hopefully this will allow you to obtain at least partial sequences for ITS.
As I know, those ITS primers are universal and useful to distungish at species level. Probably, your DNA samples are contaminated. On the other hand, how much homology you got between your sequence and BLAST hits is another issue. Anyhow, I suggest try another DNA isolation with new and clean plant material.
A paper on universal and specific ITS primers is under review. Perhaps that paper would help. You can ask MER editor for more information.
I agree with Thomas Marcussen. I had the same problems with my samples and I decided to design my own primers. At the beginning it is a lot of work, but at the end it is time- and cost saving. There are a lot of sequences in Genbank available for your alignment. You should align sequences from your target family with some fungal and bacterial sequences, and some of Chlorophyta to avoid that your designed primers will again bind on unwanted DNA.
Other plant-specific primers for the ITS 1 & 2 regions that you could try are the primers 17SE & 26SE of Sun et al 1994 - Theor. App. Genetics. These were developed in sorghum and work for us in Orchidaceae, but also some eudicots families.
I second Thomas and Christina. Also, if you wish to amplify the whole region of ITS1-5.8S-ITS2, I would suggest that you design the species specific primers that prime the 3' end of SSU 18S and 5' of domain1 in LSU 28S. We had been working on marine harmful algae and it works well almost all the time, thou with some exception for certain species which we tend to amplify the pseudogene sometimes.
The Sun primers mentioned by Nicola Flanagan work broadly for everything, but they are prone to have primer dimers, so the best strategy is that you amplify with 17SE and 26SE and use White's ITS4 and ITS5 for sequencing. Additionaly, in my experience, ITS amplifications can be unreliable if you do not add Betaine (1M final concentration) and DMSO (2% final concentration) in you ITS reaction. If with these two additives it still do not amplify, add together with them the TBT-Par formular of Samakaroon et al. 2013 in the following link http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4105354/
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