Dear Elena,
We did a fair bit of testing with polychaetes at the Biodiversity
Institute in Guelph . Mainly because we had exactly the same problems
you've described in your post: Folmer primers did not work, design of
alternative primers was an issue.
I won't say we have solved the problems 100% but we were able to make
some good progress on Polychaete barcoding. It turns out that often it
is the DNA extraction that represents the problem for further lab work
not so much the PCR itself.
The best general protocol we currently have is the following:
DNA extraction with CTAB. An example protocol utilizing 96-well plates
you can find here:
http://www.ccdb.ca/pa/ge/research/protocols/extraction (pick the plant
etc. protocol). There are also plenty of protocols on the web for
smaller scale extractions - not everyone has 96 samples available at once.
CTAB has been widely used for plants but many researchers have
discovered that it works nicely with all sort of "slimy" animals. The
idea is that this removes potential PCR inhibitors and looking at our
results in the last years it might well be true.
There is also another method to remove the suspected polysaccharides
from the extract described in this paper:
http://www.springerlink.com/content/j25wux7m21760q20/
As for the PCR we almost completely moved away from standard Folmer
primers and use Polychaete specific variants (see
http://connect.barcodeoflife.net/group/marinebarcoding/forum/topics/primer-combinations-for-marine)
and even a primer cocktail (mammal cocktail in Ivanova NV, Zemlak TS,
Hanner RH, Hebert PDN (2007) Universal primer cocktails for fish DNA
barcoding. Molecular Ecology Notes, 7, 544-548.) that initially has been
designed for vertebrates. Sometimes re-PCRing of templates that
initially did not work helped too although I see that as the very last
resort.
There is still quite some room from improvement in some groups (e.g.
some serpulids). However, recent papers have shown that there is hope :-)
Barroso R, Klautau M, Sole-Cava AM, Paiva PC (2010) Eurythoe complanata
(Polychaeta: Amphinomidae), the 'cosmopolitan' fireworm, consists of at
least three cryptic species. Marine Biology 157: 69-80
Carrera-Parra LF, Salazar-Vallejo SI (2011) Redescriptions of Eunice
filamentosa and E. denticulata and description of E. tovarae n. sp.
(Polychaeta: Eunicidae), highlighted with morphological and molecular
data. Zootaxa: 51-64
Heimeier D, Lavery S, Sewell MA (2010) Using DNA barcoding and
phylogenetics to identify Antarctic invertebrate larvae: Lessons from a
large scale study. Marine Genomics 3: 165-177
Hilário A, Johnson SB, Cunha MR, Vrijenhoek RC (2010) High diversity of
frenulates (Polychaeta: Siboglinidae) in the Gulf of Cadiz mud
volcanoes: A DNA taxonomy analysis. Deep Sea Research Part I:
Oceanographic Research Papers 57: 143-150
Luttikhuizen PC, Dekker R (2010) Pseudo-cryptic species Arenicola
defodiens and Arenicola marina (Polychaeta: Arenicolidae) in Wadden Sea,
North Sea and Skagerrak: Morphological and molecular variation. Journal
of Sea Research 63: 17-23
Schueller M (2010) Evidence for a role of bathymetry and emergence in
speciation in the genus Glycera (Glyceridae, Polychaeta) from the deep
Eastern Weddell Sea Polar Biology. Springer Berlin / Heidelberg, pp 1-16
Tovar-Hernandez MA, Carrera-Parra LF (2011) Megalomma Johansson, 1925
(Polychaeta: Sabellidae) from America and other world-wide localities,
and phylogenetic relationships within the genus. Zootaxa: 1-71
Hope that helps a bit.
Best,
Dirk
--
_______________________________________________
Dr. Dirk Steinke
Lead Scientist Marine Barcoding of Life
Biodiversity Institute of Ontario
University of Guelph
50 Stone Road West
Guelph, ON, N1G2W1
phone 1-519-824 4120 ext. 53759
fax 1-519-824-5703
email dsteinke from uoguelph.ca
