Combining insect genitalia preparation with DNAextraction

[Fabio Moretzsohn]

Dear Andrew

I have used a similar technique to digest tissue (fresh, preserved or even
dried) in situ from gastropods, mainly cypraeids, to extract the DNA and
obtain the radula and odontophore. The quality of the DNA depends (among
other factors) on the preservation of the tissue, or how fast the tissue
dried, since hydrolysis is a problem for DNA preservation. In my experience,
small specimens that dried fast can yield surprisingly good DNA, but the
best source, of course, is fresh tissue. One of the advantages of this
method is that you do not have to break the shell to extract DNA and obtain
the radula (important for taxonomy), so the technique can be used for museum
specimens without unnecessarily destroying the shell. I adapted the
technique from the following paper:

Holznagel, W. 1998. A nondestructive method for cleaning gastropod radulae
from frozen, alcohol-fixed, or dried material. American Malacological
Bulletin 14(2): 181-183.

The method uses a lysing buffer (NET) and proteinase K, and it is the best
method I know to clean radulae, making them suitable for SEM study. KOH is
commonly used to clean radulae, but it also destroys them; on the other
hand, the NET+Prot. K method does not seem to destroy the radula even after
as long as 48 hours (usually it takes only a couple of hours to digest the
tissue). I haven't tried it on insects, but it should work just as well,
give it a try.

Good luck!
Fabio

-----------------------------------------------------------
Fabio Moretzsohn, Ph.D.
Post Doctoral Research Associate
Center for Coastal Studies
Texas A&M University-Corpus Christi
6300 Ocean Drive, NRC 3208
Corpus Christi, TX 78412-5866
Phone: (361) 825-3230
Fax: (361) 825-2770
fabio at falcon.tamucc.edu

----- Original Message -----
From: "Nieukerken, E.J. van" <Nieukerken at NATURALIS.NNM.NL>
To: <TAXACOM at LISTSERV.NHM.KU.EDU>
Sent: Thursday, December 09, 2004 1:41 AM
Subject: Re: [TAXACOM] Combining insect genitalia preparation with DNA
extraction


> Yes, I have done 10 th's this way, mostly of very small moths
(Nepticulidae).
> The protocol was developed by Sonja Knölke, Andreas Segerer and others in
Munich. A paper (Knölke et al) is in press in Insect Systematics and
Evolution, as far as I know not yet published. They suggest that this should
be standard procedure!
> If you want the detailed protocol I think you should contact Andreas
Andreas.Segerer at zsm.mwn.de
>
> The results are good: enough DNA extracted and perfectly clean genitalia!
>
> Erik
>
>
>
>
> Erik J. van Nieukerken
>
> curator of Entomology (Microlepidoptera + Arachnida et al.)/
> editor Tijdschrift voor Entomologie
>
> National Museum of Natural History Naturalis
> dep. of Entomology
> PO Box 9517, 2300 RA Leiden, Netherlands
>
> [street address: Darwinweg 2, 2333 CR Leiden]
>
> direct phone: +31-71-56 87 682 (secretary ..622)
> fax:      +31-71-5687666
> e-mail:  nieukerken at nnm.nl
> http://www.naturalis.nl/nieukerken
> Tijdschrift voor Entomologie: http://www.nev.nl/tve
>
>
>  -----Original Message-----
> From:   Taxacom Discussion List [mailto:TAXACOM at LISTSERV.NHM.KU.EDU]  On
Behalf Of Andrew Mitchell
> Sent:   Thursday, December 09, 2004 2:38 AM
> To:     TAXACOM at LISTSERV.NHM.KU.EDU
> Subject:             Combining insect genitalia preparation with DNA
extraction
>
> Hello All,
>
> Has anyone experimented with using a proteinase-K/buffer solution to clear
insect abdomens for genitalia preparation, rather than using 10% KOH?  Seems
to me this would allow you to extract DNA from the gunk that one normally
throws out...  If you have tried this yourself or know of references please
e-mail me.
> Thanks,
> Andrew
> -----------------------------------------------------------------
> Andrew Mitchell
> Agricultural Scientific Collections Unit, OAI
> NSW Department of Primary Industries
> Forest Rd
> Orange NSW 2800
> AUSTRALIA
>
> Tel: +61 (0)2 6391 3848
> Fax: +61 (0)2 6391 3899
> -----------------------------------------------------------------