Additional primers?

Replies to This Discussion

Permalink Reply by Keith Seifert on October 26, 2010 at 3:06pm

At this stage, for data comparision, standardization of primers is probably not critical. But remember that standardization is a big deal in the barcoding world (practically the point of the whole thing actually), and in the end we will provide more or less a Standard Operating Protocol.

Permalink Reply by Izumi Okane on October 26, 2010 at 8:23pm

I understand the comment from Keith and agree with him. I also think that standardization of the methods is important in this project. Would it be allowed to use other primer(s) designed at the inside of target regions for a read of the full-length sequence of the regions?

Permalink Reply by Conrad Schoch on October 26, 2010 at 9:15pm

Yes, it was good of Keith to remind us of the general barcode principles. However I think getting the data in this case is priority no 1. And of course standardised primers will only apply to the gene we select for a barcode, very likely ITS. Bryn had some comments on ITS primers in an earlier post, so we may have to discuss primers issues at some later point.

Permalink Reply by Arthur Schuessler on October 30, 2010 at 6:59pm

our primers we use for Glomeromycota 200bp SSU+ITS region+800 bp LSU (1.8 kb or 1.5 kb in nested PCR) are in Krüger M, Stockinger H, Krüger C, Schüßler A (2009)
DNA-based species-level detection of arbuscular mycorrhizal fungi: one PCR primer set for all AMF.
New Phytologist 183: 212-223
http://www.genetik.biologie.uni-muenchen.de/research/schuessler/pub...

work very well, mixture instea of degenerate makes them less unspecific. not picking up Ascomycete or Basidiomycete contaminants (many AMF are cultured in open pot systems! often AMF spores are contaminated with ascos+basidios). cover ITS region and D1+D2 of LSU

see also
http://www.genetik.biologie.uni-muenchen.de/research/schuessler/pub...
Stockinger H, Krüger M, Schüßler A (2010)
DNA barcoding of arbuscular mycorrhizal fungi.
New Phytologist 187: 461-474

Permalink Reply by Conrad Schoch on November 2, 2010 at 11:55am

So it looks like ITS and LSU at least will work with this. The SSU will probably not overlap with the AFTOL sequences.

Permalink Reply by Arthur Schuessler on October 30, 2010 at 7:00pm

how long will the target region in the 5' LSU be, if included?

Permalink Reply by Conrad Schoch on November 2, 2010 at 11:53am

It should be about 900-1kb for LR0R-LR5, enough for a single sequencing run. Not sure what will work with Glomeromycota but obviously we would want as much coverage of that as possible. That said, for some of these groups we will have to take what we can get.

Permalink Reply by Arthur Schuessler on November 6, 2010 at 4:07pm

OK, this is 100 bp more (3') than our "AMF specific" (I write like this because no primer set is sure to be 100% specific) nested primers, and >100 bp less (3') than our outer primers. compatible regions, fits