Digest of responses, DNA from fish museum specimens

Bruce J. Turner fishgen at vt.edu
Thu Jul 18 09:35:20 EST 1996


The following responses were received to my query re protocols for
amplifiable DNA from preserved fish specimens.  They appear to be so
generally useful that I thought I would share them with the netlists that
generated them. This is obviously a topic of considerable interest. My
thanks to all respondents.

To: mol-evol at net.bio.net
From: arocha at ucsd.edu (Axayacatl Rocha)
Subject: Re: amplifiable DNA from museum fish specimens; methods? Date: 17
Jul 1996 14:11:00 -0700
Sender: daemon at net.bio.net
NNTP-Posting-Host: net.bio.net

Bruce, although DNA can be successfully extracted, amplified and sequenced
from formalin fixed tissues of museum specimens, my experience is that the
data is extremely noisy. I have sequenced small (ca. 350 bp) fractions of
mtDNA from fishes collected and fixed in the 50s and 60s in unbuffered
formalin and it's not good. The quality of the sequence (i.e.
chromatograms) is OK but the data per se (cyt-b) is not. Maybe someone else
has had better luck. I'm curious. Axa

At 01:15 PM 7/17/96 -0700, you wrote:
>Does anyone out there have, or know of, a reliable published protocol for
>obtaining amplifiable DNA from fish specimens in museum collections---these
(snip)
>Bruce J. Turner
>Assoc. Professor of Biology
>VPISU
>Blacksburg, VA 24061
>(540)-231-7444 (V)
>(540)-231-9307 (F)
>fishgen at vt.edu

+---------------------------------------------------------------------+ |
Axayacatl Rocha-Olivares      Phone: (619) 546-7104 |
| University of California, San Diego   Fax : (619) 546-5656 |
| Scripps Institution of Oceanography, 0208     |
| 9500 Gilman Drive     |
| La Jolla, CA 92093-0208       internet: arocha at ucsd.edu |
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Date:   Wed, 17 Jul 96 15:52:17 CDT
From: "Scott W. Herke" <SHERKE at LSUVM.SNCC.LSU.EDU> Subject:     DNA protocol
To: Bruce Turner <fishgen>
MIME-Version: 1.0
X-Mailer:       MailBook 95.01.263

Bruce,

I have a protocol that we are working on publishing. I gave it to another
squid researcher who said it worked "like gangbusters" on the formalin
preserved squid that she was doing PCR on. The protocol does not use any
toxic chemicals (e.g., CTAB, phenol, etc.). If you'd like to try the
protocol, I could mail it to you as long as you promise not to distribute
the protocol outside your lab and to let us know how well it works for you.
I say this simp ly b/c we are still in the process of finishing some tests
and writing the manuscript, so we don't want to circulate the protocol too
widely before it is actually published. One caveat I would state is that I
have had trouble duplicating the other researcher's success with
formalin-preserved squid, but I may not have used enough tissue. If you're
interested, send me your postal mailing address and I send you the
protocol.

Sincerely, Scott W. Herke (Ph.D. graduate student @ LSU) To:
mol-evol at net.bio.net
From: Adrian Spidle <spidle at fish.washington.edu> Subject: Re: amplifiable
DNA from museum fish specimens; methods? Date: Wed, 17 Jul 1996 13:55:17
-0700
NNTP-Posting-Host: pisces.fish.washington.edu Mime-Version: 1.0

You could contact Andy Shedlock at shedlock at fish.washington.edu, he has
developed a protocol to retrieve DNA from preserved angler fish.

hope that helps,

Adrian Spidle

On 17 Jul 1996, Bruce J. Turner wrote:

>Does anyone out there have, or know of, a reliable published protocol for
>obtaining amplifiable DNA from fish specimens in museum collections---these
>are routinely fixed in "formalin" and subsequently stored in ethanol or
>isopropanol. I need such a protocol to help confirm the genetic identity of a
>fish population previously thought to be extinct, but which has reappeared at
>its only known locality (there is a serious possibility here of introductions
>by humans from nearby, non-endangered populations). I haven't found anything,
>but surely some one has tackled this by now? Any help would be appreciated,
>and sorry for any duplicate mailings...

>Bruce J. Turner
>Assoc. Professor of Biology
>VPISU
>Blacksburg, VA 24061
>(540)-231-7444 (V)
>(540)-231-9307 (F)
>fishgen at vt.edu






Date: Thu, 18 Jul 1996 09:45:48 -0300 (ADT) From: Don Stewart
<dstewart at is.dal.ca>
To: fishgen
Subject: Amps and formalin
MIME-Version: 1.0

DEar Dr. Turner,
At the Evolution meetings in Edinburgh last September, I saw a poster
devoted to this subject: DNA amplifications from specimens fixed in
unbuffered formaldehyde and fixed in ethanol. Apparently the authors had
success in amplifying fragments up to 500 bp in length. Their address is:
Anne-Marie Vachot and Monique Monnerot
CNRS
Centre de Genetique Moleculaire
F-91198 Gif sur Yvette cedex,
France

I could fax you the abstract but it does not contain any of their secrets.
Good luck,

Don Stewart


************************
Dr. Don Stewart
Dept. of Biology
Dalhousie University
Halifax, N.S.
B3H 4J1
Tel. (902) 494-3579
Fax. (902) 494-3736
e-mail dstewart at is.dal.ca
*************************
Date: Thu, 18 Jul 1996 05:38:42 -0700 (PDT) X-Sender: fzmay at peseta.ucdavis.edu
Mime-Version: 1.0
To: fishgen
From: bpmay at ucdavis.edu (Bernie May)
Subject: museum protocols
X-Mailer: <Windows Eudora Version 2.0.2>

Bruce-
I don't have any help for you but I do have a student interested in such
material. He will be working on skates, rays, or sawfish from museum
specimens. Please forward any good e-mail replies to me. Hope all goes well
with you.
-Bernie

----------------------------------
Bernie May, Associate Research Biologist Director, Genomic Variation Laboratory
Deptartment of Animal Science
2237 Meyer Hall (office) 2403 (lab)
University of California
Davis, CA 95616
office (916) 754-8123
FAX (916) 752-0175
lab (916) 752-6351
E-mail bpmay at ucdavis.edu

For a copy of the latest "Genes in Populations" for analyzing Mendelian
data visit the following site.
http://animalscience.ucdavis.edu/software/may.htm

Date: Thu, 18 Jul 1996 12:04:27 +0100 (BST) From: Iain M Matthews
<imm at st-andrews.ac.uk> X-Sender: imm at bute
To: fishgen
MIME-Version: 1.0

Dear Bruce,
I had this message passed on to me by Jeff Graves here in St Andrews. There
is a protocol that I have used for extraction of DNA from preserved
specimens.
The protocol I used was taken from :

Taggart, J.B., Hynes, R.A., Prodohl, P.A. and Ferguson, A. (1992) A
simplified protocol for routine total DNA isolation from salmonid fishes.
Journal of Fish Biology. 40 : 963-965.

I use the protocol for paternity analysis poeciliids and I have heard of it
being used for a number of other species. The paper mentions the various
steps to get round the problems with preserved samples.

Best of luck. If you have any other questions, feel free to get in touch.

Iain Matthews.
School of Biological and Medical Sciences. University of St Andrews.
St Andrews.
Scotland. U.K.

Tel : +44 1334 476161 Ext 3220
Fax : +44 1334 463600

Date: Thu, 18 Jul 96 10:43:39 PDT
From: Monique Monnerot <Monique.Monnerot at cgm.cnrs-gif.fr> Subject: formalin
To: fishgen
X-Mailer: Chameleon - TCP/IP for Windows by NetManage, Inc. MIME-Version: 1.0

Bruce
I too have been working on extracting and amplifying DNA from
formalin-fixed specimens (frogs). I succeed in amplifying fragments up to
450 bp lenght using a new protocol I designed. I have a paper accepted for
publication in the first issues of Ancient Biomolecules (a new journal)
which had to appear in february 96. Because of troubles from the editor,
this issue is supposed to arrive in august 96.
would you like to get a copy of this paper by regular mail and/or a copy by
the recipe by email?
sincerely
Anne-marie Vachot



------------------------------------------------------- | Monique MONNEROT
Tel: (33-1) 69824358
| Centre de Genetique Moleculaire Fax: (33-1) 69074973 | CNRS
| 91198 Gif sur Yvette Cedex
| France        E-mail: Monnerot at cgm.cnrs-gif.fr
-------------------------------------------------------

X-Sender: jja at mailserver.nhm.ac.uk
Mime-Version: 1.0
Date: Thu, 18 Jul 1996 07:58:36 +0100
To: fishgen (Bruce J. Turner)
From: J.Austin at nhm.ac.uk (Jeremy Austin) Subject: Re: amplifiable DNA from
museum fish specimens; methods?

>Does anyone out there have, or know of, a reliable published protocol for
>obtaining amplifiable DNA from fish specimens in museum collections---these
>are routinely fixed in "formalin" and subsequently stored in ethanol or
>isopropanol. I need such a protocol to help confirm the genetic identity of a
>fish population previously thought to be extinct, but which has reappeared at
>its only known locality (there is a serious possibility here of introductions
>by humans from nearby, non-endangered populations). I haven't found anything,
>but surely some one has tackled this by now? Any help would be appreciated,
>and sorry for any duplicate mailings...

>Bruce J. Turner
>Assoc. Professor of Biology
>VPISU
>Blacksburg, VA 24061
>(540)-231-7444 (V)
>(540)-231-9307 (F)
>fishgen at vt.edu


point your inter-library loan forms at:

Shiozawa, D. K., J. Kudo, R. P. Evans, S. R. Woodward, and R. N. Williams.
1992. DNA extraction from preserved trout tissues. Great Basin Naturalist,
52:29-34.

They calimed to have gotten DNA from formalin fixed tissues. Essentially it
involved several 24hr or greater "soaks" of the tissue to remove any traces
of formalin and/or ethanol, followed by extended digestion with Prot K and
SDS, followed by phenol:chloroform cleanup. The trick I think was the long
soaks and digestion times. This technique seemed to work on a whole range
of fish tissue including formalin fixed fins.

Hope this helps!

Jeremy Austin



--------------------------------------------------------------------- Dr
Jeremy Austin  INTERNET: J.Austin at nhm.ac.uk
Department of Palaeontology     Phone: +44 (0)171 938 9131
and Department of Zoology       Fax: +44 (0)171 938 8754
The Natural History Museum,
Cromwell Road, London SW7 5BD, UK

"I believe in coyotes and time as an abstract Explain the change, the
difference between What you want and what you need, there's the key Your
adventure for today, what do you do Between the horns of the day?"
R.E.M

---------------------------------------------------------------------




X-Sender: fishgen at mail.vt.edu
X-Mailer: Windows Eudora Light Version 1.5.2 Mime-Version: 1.0
Date: Mon, 15 Jul 1996 01:52:28 +0600
To: fishgen
From: "Bruce J. Turner" <fishgen at vt.edu> Subject: amplifiable DNA from
museum fish specimens; methods?

>To: mol-evol at net.bio.net
>From: shedlock at NATURE.BERKELEY.EDU ("Andrew M. Shedlock") Subject: amplifiable
>DNA from museum fish specimens; methods? Date: 17 Jul 1996 16:02:27 -0700
>Sender: daemon at net.bio.net
>NNTP-Posting-Host: net.bio.net

>Dear Bruce and others to whom it may concern,

>I have a paper in review for BioTechniques right now on this exact issue for
>which I'm expecting to receive a verdict any day. Along with colleagues at the
>Scripps Institution and the University of Washington, I developed a modified
>extraction/PCR protocol that worked on a variety of deep-sea fish specimens,
>some of which had been formalin-fixed almost a century ago. It's far from the
>first attempt to get DNA data from museum specimens, but, in my humble
>opinion, it's a valuable starting point and has plenty of room for
>optimization with other formalin-fixed, fluid-preserved material.

>I'd be willing to share elements of the recipe with you or anyone who is
>interested and willing to acknowledge the (as of today) yet unpublished work
>in any manuscripts submitted prior to it's acceptance for BioTechniques.

>All the best --Andy
>.....................................................................
..T...G...A...C......................
>Andrew M. Shedlock     ..T.G...A...C... Molecular Ecology..
>Univ. of California at Berkeley ...T...A...C...... & Systematics ... Dept.
>E.S.P.M.    ....T.A...C.........................
>201 Wellman Hall       .....T...C... Office: (510) 642-3989
>Berkeley, CA 94720     ......T.C.... Lab: (510) 642-7410
>shedlock at nature.berkeley.edu   .......T..... Fax: (510) 642-0477
>........................................T............................



>---------- Forwarded message ---------- Date: Wed, 17 Jul 1996 16:02:27 -0400
>From: "Bruce J. Turner" <fishgen at vt.edu> To: molecular-evolution at net.bio.net
>Cc: systbiol at sonofsun.sdsu.edu
>Subject: amplifiable DNA from museum fish specimens; methods?

>Does anyone out there have, or know of, a reliable published protocol for
>obtaining amplifiable DNA from fish specimens in museum collections---these
>are routinely fixed in "formalin" and subsequently stored in ethanol or
>isopropanol. I need such a protocol to help confirm the genetic identity of a
>fish population previously thought to be extinct, but which has reappeared at
>its only known locality (there is a serious possibility here of introductions
>by humans from nearby, non-endangered populations). I haven't found anything,
>but surely some one has tackled this by now? Any help would be appreciated,
>and sorry for any duplicate mailings...

>Bruce J. Turner
>Assoc. Professor of Biology
>VPISU
>Blacksburg, VA 24061
>(540)-231-7444 (V)
>(540)-231-9307 (F)
>fishgen at vt.edu







Mime-Version: 1.0
Date: Wed, 17 Jul 1996 20:37:53 -0800
To: fishgen (Bruce J. Turner)
From: adam at darwin.ucsc.edu (adam chippindale) Subject: Re: amplifiable DNA
from museum fish specimens; methods?

Bruce,

Try my brother, Paul (paulc at albert.uta.edu). He might have an answer, but
could probably put you on the right track in any event.

Adam

__________________________

ADAM CHIPPINDALE
Dept. of Biology @ N.S. IV
University of California
Santa Cruz, CA 95064 USA
FAX: 408 459 4882
PH: 408 459 5147
__________________________


Date: Thu, 18 Jul 1996 09:13:26 +0700 (GMT+0700) From: Pamela Gail Gregory
- fr <frpgo at mahidol.ac.th> Reply-To: Pamela Gail Gregory - fr
<frpgo at mahidol.ac.th> To: "Bruce J. Turner" <fishgen>
Subject: Re: amplifiable DNA from museum fish specimens; methods?
MIME-Version: 1.0


Bruce,

I know of a paper, but of course I can't remember the reference. My friend
Connie, however, has the paper. You may e-mail her directly at
ckeelerf at nmsu.edu. She does research on fish as well and I think you both
should communicate.

Best wishes!

Pamela


On Wed, 17 Jul 1996, Bruce J. Turner wrote:

>Does anyone out there have, or know of, a reliable published protocol for
>obtaining amplifiable DNA from fish specimens in museum collections---these
>are routinely fixed in "formalin" and subsequently stored in ethanol or
>isopropanol. I need such a protocol to help confirm the genetic identity of a
>fish population previously thought to be extinct, but which has reappeared at
>its only known locality (there is a serious possibility here of introductions
>by humans from nearby, non-endangered populations). I haven't found anything,
>but surely some one has tackled this by now? Any help would be appreciated,
>and sorry for any duplicate mailings...

>Bruce J. Turner
>Assoc. Professor of Biology
>VPISU
>Blacksburg, VA 24061
>(540)-231-7444 (V)
>(540)-231-9307 (F)
>fishgen at vt.edu









^       MMM     MMM UU  UU      ^
^^^     MM M    M MM UU UU      ^^^
^^^^^^^ MM M M MM UU    UU ^^^^^^^
^^^     MM M MM UU UUU  ^^^
^       MM      MM      UUU U   ^


Pamela G. Gregory       e-mail: frpgo at mahidol.ac.th
Visiting Professor      Tel & Fax: 662-247-7051
Department of Biology   Tel (off): 662-246-0063
Faculty of Science      ext. 2506, 2509
Mahidol University
Rama 6 Road     ^
Bangkok 10400   ^^^
^^^^^^^
^^^
^



Date: Thu, 18 Jul 1996 09:44:32 -0400 (EDT) From: Thomas D Kocher
<tdk at hopper.unh.edu> To: Bruce Turner <fishgen>
Cc: Scott C France <scf at hopper.unh.edu>
Subject: formalin fixed fish
Mime-Version: 1.0


Hi Bruce,

I have a postdoc, Scott France, who has made an extensive study of
formalin-fixed, ethanol-preserved amphipod specimens from the deep-sea.
There is some hope, if your fish were preserved in the last 20 years. He's
out of town now, but I'll have him forward you some info when he returns.
I'll just add the observation that we have much better luck extracting good
DNA from fins than livers - when ethanol-preserved (no formalin step). I
think this is because nucleases work in the liver for a while before the
ethanol gets in. Not sure whether fins will be better for formalin-fixed
stuff.

-Tom Kocher
From: "Eric Taylor" <etaylor at zoology.ubc.ca> X-Host-Id: light.zoology.ubc.ca
Date: Wed, 17 Jul 96 17:47:49 PDT
To: fishgen
Subject: Re: amplifiable DNA from museum fish specimens; methods?

Hi. There is a paper in the Great Basin Naturalist 52:29-34 by Shiozawa et
al. 1992 that gives a protocol. It didn't work as well for me, but I
skimmped on a few steps.

Eric Taylor
Assistant Professor
Univ. of B.C.
Dept. of Zoology
Vancouver, Canada
Date: Wed, 17 Jul 1996 20:29:09 -0400 (EDT) X-Sender:
mds25 at postoffice4.mail.cornell.edu Mime-Version: 1.0
To: fishgen (Bruce J. Turner)
From: mds25 at cornell.edu (malcolm schug)
Subject: Re: amplifiable DNA from museum fish specimens; methods?

Bruce -

You might try asking Paul Fuerst or his postdoc Greg Booton at Ohio State
if you have no luck from your query on the evol. directory. I know they
have worked on this problem with museum specimens of sturgeon.

you can e-mail them at:

fuerst.1 at osu.edu
booton.1 at osu.edu

Good luck!

Malcolm Schug




>Does anyone out there have, or know of, a reliable published protocol for
>obtaining amplifiable DNA from fish specimens in museum collections---these
>are routinely fixed in "formalin" and subsequently stored in ethanol or
>isopropanol. I need such a protocol to help confirm the genetic identity of a
>fish population previously thought to be extinct, but which has reappeared at
>its only known locality (there is a serious possibility here of introductions
>by humans from nearby, non-endangered populations). I haven't found anything,
>but surely some one has tackled this by now? Any help would be appreciated,
>and sorry for any duplicate mailings...

>Bruce J. Turner
>Assoc. Professor of Biology
>VPISU
>Blacksburg, VA 24061
>(540)-231-7444 (V)
>(540)-231-9307 (F)
>fishgen at vt.edu

Malcolm Schug
Postdoctoral Research Associate
Section of Genetics and Developement
Cornell University
403 Biotechnology Bldg
Ithaca, NY 14853-2703

Phone 607-254-4839
e-mail mds25 at cornell.edu


Date: Wed, 17 Jul 1996 14:22:41 -1000
From: Steven M Carr <carr at hawaii.edu>
X-Sender: carr at uhunix3
To: "Bruce J. Turner" <fishgen>
Subject: Re: amplifiable DNA from museum fish specimens; methods?
MIME-Version: 1.0

Bruce

I reviewed an MS for TAFS on this very subject a few years back - I know
that I saw it in the journal, but I can't remember the author or title
(some guy at U Texas). I wasn't overly impressed with the science reported,
but it was a specific method for formalin-fixed fish, using scale
epidermis. Must be since 1993 - sorry not to be more specific.

Hope this helps

Steve

***************************************************************************
Steven M. Carr
Dept. of Biology        "Provehito in Altum"
Memorial University of Newfoundland
St. John's NF A1B 3X9
CANADA

>From 01 May - 05 July 1996 at:

Kewalo Marine Laboratory        Today's weather in Honolulu:
University of Hawaii    High in the 80s, low in the 70s,
41 Ahui St.     Mostly sunny, tradewinds 15-20 mph
Honolulu HI 96813       Windward & Mauka showers
"Lucky you live Hawaii!"

(808) 539-7311 office & lab / -7300 messages / 599-4817 FAX carr at hawaii.edu
***************************************************************************


On Wed, 17 Jul 1996, Bruce J. Turner wrote:

>Does anyone out there have, or know of, a reliable published protocol for
>obtaining amplifiable DNA from fish specimens in museum collections---these
>are routinely fixed in "formalin" and subsequently stored in ethanol or
>isopropanol. I need such a protocol to help confirm the genetic identity of a
>fish population previously thought to be extinct, but which has reappeared at
>its only known locality (there is a serious possibility here of introductions
>by humans from nearby, non-endangered populations). I haven't found anything,
>but surely some one has tackled this by now? Any help would be appreciated,
>and sorry for any duplicate mailings...

>Bruce J. Turner
>Assoc. Professor of Biology
>VPISU
>Blacksburg, VA 24061
>(540)-231-7444 (V)
>(540)-231-9307 (F)
>fishgen at vt.edu






Date: Wed, 17 Jul 1996 17:14:10 -0700 (PDT) X-Sender: arochaol at sdcc3.ucsd.edu
X-Mailer: Windows Eudora Light Version 1.5.2 Mime-Version: 1.0
To: fishgen (Bruce J. Turner)
From: Axayacatl Rocha <arocha at ucsd.edu>
Subject: Re: amplifiable DNA from museum fish specimens; methods?

Perhaps I should have talked about "preservation artifacts" instead of
noise. The sequence per se was not noisy (i.e. the base calls were
unequivocal) as the PCR and sequencing reactions went OK, but when you
compared the data with those of conspecific fresh individuals the amount of
nuc-changes incrased + 10x. I could reliable assess the presence of
artifacts when comparing "fresh" and "fixed" sequences because I was
looking at a coding region, I think this would have been more difficult to
assess in the control region. If you're still interested I'll gladly send
you what I have. Please keep me posted on any success stories you might
hear on the back channel. Best of lucks. Axa

P.S. Yes the DNA was obviously degraded, as far as the minigel image goes.
I was only successful at getting DNA from liver, not from muscle.

At 05:39 PM 7/17/96 -0400, you wrote:
>Thanks, Axa: We are going after a piece of the D-loop of about the same size:
>Do you mean that the sequence was noisy or that the DNA was degraded in some
>other obvious way?

>Bruce J. Turner
>Assoc. Professor of Biology
>VPISU
>Blacksburg, VA 24061
>(540)-231-7444 (V)
>(540)-231-9307 (F)
>fishgen at vt.edu





+---------------------------------------------------------------------+ |
Axayacatl Rocha-Olivares      Phone: (619) 546-7104 |
| University of California, San Diego   Fax : (619) 546-5656 |
| Scripps Institution of Oceanography, 0208     |
| 9500 Gilman Drive     |
| La Jolla, CA 92093-0208       internet: arocha at ucsd.edu |
+---------------------------------------------------------------------+


                        Bruce J. Turner
                        Assoc. Professor of Biology
                        VPISU
                        Blacksburg, VA 24061
                        (540)-231-7444 (V)
                        (540)-231-9307 (F)
                        fishgen at vt.edu






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