Below is a compilation of some of the
responses to an appeal for protocols
on DNA extraction form non buffered
formalin.
The appeal prompted lots of
interesting responses some of which
are listed below. Others are partially
listed with the contact addresses
removed (I have contacted them and
will post their replies if they agree)
other respondents mention
unpublished protocols for 600bp+ but
do not at present wish the information
to be freely available until they have
submitted. I will make available all
comments and protocols that the
authors do not explicitly do not wish
me to do so.
I seams to me that there is a lot of
interest in this area and exchanged
information could really useful. I am
not very computer literate and am not
sure of the best way that can be set up
so discussion can continue. Any
suggestions????
I have added a few comments please
don't let this give the impression that I
am some kind of expert on this
subject.
I have done a fairly comprehensive
literature search and have included
few of the more generally relevant
papers. There are several hundred
papers on amplification from medical
paraffin embedded specimens but as
phosphate buffered Formalin was
used they are not in the vast majority
of cases particularly interesting.
By the way I am not a Doctor as some
people thought, I am a Ph.D. student.
My project is on the
evolutionary/population genetics of a
Family of reef fish. I am hoping to
use samples to amplify mitochondrial
DNA by PCR and then to directly
sequence (most probably after a
purification step).
I have sent identical copies of this to
all respondents (and other people that
may be intrested) and hope that
everybody will contact anybody they
wish to. I would be good if everybody
could keep others informed of
successful or equally failed
approaches.
Date: Fri, 5 May 1995 11:01:08 -0600
To: R.Guy.Reeves at ncl.ac.uk (Guy Reeves)
From: pswhite at noble.org (P. Scott White)
Subject: Formalin and DNA isolation
Cc: pswhite at noble.org
X-PMFLAGS: 34078848
Guy,
I read your request for info on the bionet,
but thought I would
reply individually. I have examined extraction
protocols and adapted some
for such applications, and worked out a simple
method that seems to work on
most any tissue, including formalin preserved
specimens (although with much
lower efficiency). I too felt that the DNA was
still in the tissue, and
that it was more a matter of getting it out than it
being chopped up. In a
methods review I demonstrated the size of DNA
isolated from such tissue to
be as high as 23 kb or more by end-labeling it
and running it out on a
vertical agarose gel. The figure is in the paper,
but the editor had me
remove a gel that showed the PCR product from
the sample, which was about
330 bp. The reference is as follows:
White, P.S. and L.D. Densmore. Mitochondrial
DNA. In Molecular Genetic
Analysis of Populations: A Practical Approach.
(A. R. Hoelzel, ed), IRL
Press, Oxford (1992) pp. 29-58.
If you have problems finding this, let me know
and I will e-mail the
protocol. My guess is that there may still be
some PCR inhibitors in many
of these samples, but methods for getting rid of
those have been recently
developed (see NAR 23:881-882). I also think
that a precipitation using
2-butoxyethanol as described in a paper by R.
Manning (Analytical
Biochemistry 195:45-50. 1991) will do the trick
better than EtOH precip,
but I have never tried it (We use it here to get rid
of phenolics and
carbohydrates from plant tissue extracts). Good
luck, and let me know how
it works.
Sincerely,
P. Scott White
S.R. Noble Foundation, Inc.
Ardmore, OK 73402 USA
-- End --
Date: Fri, 5 May 1995 07:22:41 -0500
From: Mary Curtis <curtis1 at maxwell.iia2.org>
Message-Id:
<199505051222.HAA13873 at maxwell.iia2.org>
To: R.Guy.Reeves at ncl.ac.uk (Guy Reeves)
Subject: Re: DNA from FORMALIN fixed
SPECIMINES
Newsgroups: bionet.molbio.evolution
X-Newsreader: TIN [version 1.2 PL2]
X-PMFLAGS: 33554560
I know of someone at the University of
Michigan who has tried and
succeeded in getting some DNA from formalin
specimens, although I think
the formalin may have been phosphate buffered.
I've been getting intact
large pieces of DNA (>800 kb) from dried skin
using an ion-resin exchange
buffer (Chelex)--some other folks in the midwest
USA are using Dynabeads
(magnetic beads) that can be purchased
commercially. I think Stratagene
makes a kit that works well--I can get the product
name if you are
interested.
----------------------------------------------------------
I have cut out a bit here.
I am contacting the person she mentions here and
hope she willl allow the response to be posted
----------------------------------------------------------
PS: we've all been working on fishes...
Mary K. Burnham Curtis
National Biological Service-Great Lakes Science
Center
Mary_Curtis at nbs.gov or
curtis1 at maxwell.iia2.org
-- End --
Date: Thu, 04 May 1995 12:57:44 +0000
From: gbga13 at udcf.gla.ac.uk (B.L.Cohen)
To: Guy Reeves <R.Guy.Reeves at ncl.ac.uk>
Cc: gbga13 at udcf.gla.ac.uk
Subject: Re: DNA from FORMALIN fixed
SPECIMINES
References: <3nqoij$o0s at whitbeck.ncl.ac.uk>
Organization: Genetics, University of Glasgow
X-PMFLAGS: 33554560
The most likely explanation I have seen for the
failure of formalin-fixed
tissues is that DNA is not fixed by crosslinking
and is free to diffuse out
so long as only formalin is present. Thus a quick
dip in it followed by
ethanol is reputed to be OK.
My interest level is high because many museum
specuimens of brachiopods
have been fixed in formalin. I have never been
able to PCR from their DNA
preps.
----------------------------------------------------------
It is my impression from the litrature that this is
unlikley to be the correct explanation.
Guy
----------------------------------------------------------
-- End --
Date: Wed, 3 May 1995 17:58:41 -0400 (EDT)
From: Scott C France <scf at hopper.unh.edu>
To: R.Guy.Reeves at ncl.ac.uk
Subject: re: DNA from formalin-fixed specimens
Message-Id:
<Pine.OSF.3.91a.950503175646.3841A-
100000 at hopper.unh.edu>
Mime-Version: 1.0
Guy,
I have been working on the problem of using
formalin-fixed tissues in
PCR-based analyses for about a year. My
interests are specifically on
deep-sea invertebrates, most of which are small
and difficult to collect,
but for which many historical collections exist -
hence the need for
working with formalin-fixed specimens.
I can't tell you very much at this point (not
because it is secret, but
because I don't have the answers yet). To date, I
have been able to
amplify fragments up to about 350 bp long from
material 20 years old (not
20 years in formalin though - the material was
transferred into ethanol
at some point), and by using different sets of
primers, have been able to
overlap them to provide sequence up to 500 bp
long (haven't had primers
made to extend beyond that point). Although I
can't say for sure, I
suspect the formalin was phosphate buffered
(marine samples usually
are). The extractions I have been doing are
derived from a couple of
papers which used formalin-fixed material:
Shiozawa, D.K., Kudo, J.,
Evans, R.P., Woodward, S.R., and Williams,
R.N. (1992). DNA extraction
from preserved trout tissues. Great Basin
Naturalist 52: 29-34; and Goelz
et al (1985) which is cited in the reference you
gave.
---------------------------------------------
I knew of the prottocol but figured that
it cost over £4.00 just in protienase K per
tube
---------------------------------------------
I was not aware of the reference you gave (Lab.
Invest.) - thanks. I
have several of the "chemical journal" papers
cited in this paper and
have wanted to try modifications of pH and
temperature to see if that
would help reverse the cross-linking. I should be
beginning those
experiments sometime later this month and
through the summer. I would
like to hear what other replies you get on this
subject. Keep in touch.
Scott France scf at christa.unh.edu
Department of Zoology
University of New Hampshire
Durham, NH 03824 USA
----------------------------------------------------------
-------------
-- End --
Date: Wed, 3 May 1995 17:47:34 -0700
Message-Id:
<199505040047.RAA28952 at ix5.ix.netcom.co
m>
From: dpbijb at ix.netcom.com (David Bick)
Subject: Formalin fixed specimens
To: R.Guy.Reeves at ncl.ac.uk
X-PMFLAGS: 33554560
I too have an interest in PCR from formalin fixed
specimens. I am
using chelex with some sucess although not for
long amplification. It
seems to work for PCR products <400 bp. but
not on all samples. If you
come across a better approach I would be
interested.
- David Bick
-- End --
Date: Wed, 3 May 1995 15:34:38 -0400
X-Sender: fishgen at mail.vt.edu
Mime-Version: 1.0
Content-Type: multipart/mixed;
boundary="========================_1
5643116==_"
To: Guy Reeves <R.Guy.Reeves at ncl.ac.uk>
From: fishgen at vt.edu (Bruce J. Turner)
Subject: Re: DNA from FORMALIN fixed
SPECIMINES
Here's a reference I downloaded from the
reagents and methods group some
months ago. We are also interested in doing this,
but haven't gotten
around to it yet. The "Tween" idea is
particularly intriguing, and is in
line with your thinking on the issue. We have
some formalin-fixed fish
specimens now being transferred into ethanol (a
la standard museum
specimens) and plan to try to isolate the DNA
after about a month. Then we
will try PCR of a d loop primer we are fond of,
and perhaps some other
things. I suspect that many different groups are
trying to amplify
formalin-fixed specimens, and it might be a real
contribution if you could
collate all the responses you get and put them out
on the net.
-----------------------------------
HOw do you think is the best way
to put them ion the net?
-----------------------------------
--
========================_15643116==
_
Content-Type: text/plain; name="RE-
_PCR_of_formalin_fixed_tissu"; charset="us-
ascii"
Content-Disposition: attachment; filename="RE-
_PCR_of_formalin_fixed_tissu"
To: methods-and-reagents at net.bio.net
From: slebos at amc.uva.nl
Subject: RE: PCR of formalin fixed tissues Date:
Fri, 2 Dec 94 16:26:47 GMT
Nntp-Posting-Host: amccca.amc.uva.nl
In Article <24NOV94.08413405 at admin3>
chelack at admin3 writes:
>I am also trying to set up PCR methods for
formalin fixed tissue sections and
>am concerned that the length of time the tissue
was in formalin greatly
>affects the ability to amplify products. In
general I am deparaffinizing the
>sections followed by freeze/thaw fracturing of
the tissue and lengthy
>proteinase K digestion then phenol chloroform
extraction. We are working on
>the identification of M. bovis in elk samples.
The samples we have are fixed
>for an unknown length of time and actually may
vary in fixation time among the
>samples. So far I am able to obtain product
from 4/7 samples which leads me to
>believe that fixation time may be the culprit.
Does anyone else out there have
>any other suggestions regarding this, or any
other secret tricks they wish to
>share?
>Regards
>BJC Never worry about others trying to steal
>your ideas, if they are any good at all
>you will have to ram them down their throats.
We have been amplifying DNA from fixed
tissues for quite some time. The
best protocol we have is actually very simple. It
was described in Diagn.
Mol. Pathol. 1, 136-141 (1992). The concept is
that you incubate the tissue
in prot K and a non-ionic detergent like Tween
(in which Taq amplifies
perfectly fine), incubate for one night at 56C to
reverse the formalin
fixation, and use the resulting supernatant
directly for PCR. This has
worked on over 90% of the cases we tried.
Good Luck,
Rob Slebos
University of Amsterdam Medical Center
Dept. Pathology
Meibergdreef 9
1105AZ Amsterdam
The Netherlands
E-mail: slebos at amc.uva.nl
--
========================_15643116==
_--
-- End --
From: wfischer at bio.indiana.edu (Will Fischer)
Newsgroups: bionet.molbio.evolution
Subject: Re: DNA from FORMALIN fixed
SPECIMINES
Date: 5 May 1995 16:54:14 GMT
Organization: Biology, Indiana University -
Bloomington
Lines: 35
Guy Reeves (R.Guy.Reeves at ncl.ac.uk) wrote:
Formaldehyde has been used for DNA-protein
cross-linking because it is
reversible (so claim these authors):
Orlando, V. and Paro, R. (1993) Mapping
polycomb-repressed domains in
the bithorax complex using in vivo formaldehyde
cross-linked chromatin.
Cell 75:1187-1198 (Dec 17 1993).
They got the cross-links off by SDS-proteinase
K treatment... but I
suspect that the cross-linking they dealt with
wasn't as extensive as
you might find in preserved tissue. You could
also check out the
following:
Solomon, M.J. and Varshavsky, A. (1985)
Formaldehyde-mediated
DNA-protein crosslinking: a probe for in vivo
chromatin structures.
Proc. Natl. Acad. Sci. USA 82:6470-6474.
-- Good luck.
_______________________________________
_____________________
Will Fischer
wfischer at indiana.edu
Department of Biology Lab: 812-
855-2549
Jordan Hall
Indiana University FAX: 812-
855-6705
Bloomington, Indiana 47405 USA
Date sent: Mon, 15 May 1995 14:33:18
+0100
From: inclaes at alpha1.bids.ac.uk
To:
R.Guy.Reeves%ncl.ac.uk at away.bids.ac.uk
Subject: citing paabo 1990
Copyright 1995, Institute for Scientific
Information Inc.
Database : Science Citation Index
Refreences
TI- FORMALIN-INDUCED INFIDELITY IN
PCR-AMPLIFIED DNA FRAGMENTS
AU- DEGIORGI, C;SIALER, MF;LAMBERTI,
F
NA- UNIV BARI,DIPARTMENTO BIOCHIM
& BIOL MOLEC,VIA AMENDOLA 165-A,I-
70126
BARI,ITALY
CNR,IST NEMATOL AGRARIA,I-70126
BARI,ITALY
JN- MOLECULAR AND CELLULAR
PROBES
PY- 1994
VO- 8
NO- 6
PG- 459-462
AB- A 643-nucleotide-long fragment of rDNA
gene was amplified by PCR in the
nematode worm Caenorhabditis elegans.
When the experiments were
performed by using samples fixed in formalin,
artefacts were detected.
While the size of the amplified fragment
resulted unaffected, very
striking differences were seen in the nucleotide
sequences of the
amplified fragments. Furthermore, in many
cases, the PCR reaction
failed completely. The results obtained might
warn of potential
problems, especially when the amount of
DNA to be amplified is scarce.
----------------------------------------------------------
------
I haven't got a copy of this paper yet but it apears
to be intresting
----------------------------------------------------------
------
TI- DNA EXTRACTION BY SONICATION -
A COMPARISON OF FRESH, FROZEN,
AND
PARAFFIN-EMBEDDED TISSUES
EXTRACTED FOR USE IN POLYMERASE
CHAIN-REACTION ASSAYS
AU- HELLER, MJ;ROBINSON,
RA;BURGART, LJ;TENEYCK, CJ;WILKE,
WW
NA- UNIV IOWA,COLL MED,DEPT
PATHOL,DIV SURG PATHOL,IOWA
CITY,IA,52242
JN- MODERN PATHOLOGY
PY- 1992
VO- 5
NO- 2
PG- 203-206
AB- DNA extraction from fixed tissues can be
the most laborious and complex
step in amplifying DNA by the polymerase
chain reaction (PCR). We have
previously reported a rapid and efficient
method for extracting DNA by
the use of sonication and glass beads. We have
extended our experiences
with this technique using fresh, frozen, and
formalin-fixed
paraffin-embedded tissues with and without
the use of glass beads and
report their results. Multiple tissue types were
obtained at autopsy or
as part of a surgical specimen. DNA was
extracted from identical tissue
when the sample was fresh, frozen, or
formalin-fixed paraffin-embedded.
Our results indicate that in most instances the
sonication technique,
which takes only 30 min from start to finish,
can rapidly extract
fresh, frozen, or formalin-fixed paraffin-
embedded tissue and is
superior to other rapid extraction techniques in
terms of quality and
quantity of DNA. It is much more rapid than
those techniques that use
long digestion periods. This technique will be
of great value to those
investigators extracting DNA for polymerase
chain reaction assays.
-----------------------------
quite a neat aproach only takes
30 mins
-------------------------------
TI- PCR AMPLIFICATION OF 40-YEAR-
OLD PARAFFIN-EMBEDDED TUMOR-
TISSUES -
COMPARISON OF 4 DIFFERENT DNA
EXTRACTION AND PURIFICATION
METHODS
AU- WANG, WG;KUMAR, P;SCHWARZ,
M;MALONE, G;HAWORTH, A;KUMAR, S
NA- MANCHESTER METROPOLITAN
UNIV,DEPT BIOL SCI,MANCHESTER M1
5GD,LANCS,ENGLAND
MANCHESTER METROPOLITAN
UNIV,DEPT BIOL SCI,MANCHESTER M1
5GD,LANCS,ENGLAND
ROYAL MANCHESTER CHILDRENS
HOSP,REG MOLEC GENET
LAB,MANCHESTER M27
4HA,LANCS,ENGLAND
CHRISTIE HOSP & HOLT RADIUM
INST,CLIN RES LABS,TUMOR BIOL
LAB,MANCHESTER,LANCS,ENGLAND
JN- INTERNATIONAL JOURNAL OF
ONCOLOGY
PY- 1994
VO- 5
NO- 3
PG- 453-457
AB- Four different methods of DNA extraction
from formalin-fixed,
paraffin-embedded tissues were compared for
their ability to produce
DNA suitable as a template for polymerase
chain reaction (PCR). Seven
paraffin-embedded rhabdomyosarcoma tissue
blocks from the 1950s and one
from 1960 were treated with the following
extraction methods: 1.
Proteinase K digestion and phenol/chloroform
extraction; 2. Proteinase
K digestion followed by boiling to inactivate
the enzyme; 3. Proteinase
K digestion, addition of Chelex-100, followed
by boiling; and 4.
Proteinase K digestion and Prep-A-Gene
purification. DNA extracted by
methods 1 and 2 was degraded, but DNA of
high molecular weight was
recovered in every sample extracted by
methods 3 and 4 - even though
some degradation was observed. Extracted
DNA was used as a template for
PCR amplification of exon 4 of the PAX-3
gene. PCR was successful in 7
out of 8 samples prepared using methods 3
and 4, producing levels of
amplified product equivalent to those obtained
with control DNA
obtained from fresh lymphocytes. Only very
weak products were found in
samples prepared by methods 1 (2 out of 8)
and 2 (4 out of 8). These
results indicate that chelation of polyvalent
ions (Chelex-100 method)
or obviating the need for boiling (Prep-A-
Gene) may protect DNA during
extraction.
-------------------------------
haven't got a copy yet
-------------------------------
--------------------------------
The 3 papers below are the most
interesting (I think) and apear to confirm each
other's results. The Japanese ones are
especially intersting for anybody intrested
formalin DNA (loads of references). They
peform the kind of experiments that most
peolpe would do given an infinite amount
of free time and enthuseasm. Data on
unbuffered
formailin and chalk adjusted formailin (a not
uncommon
proceedure for some kinds of biological
specimines),
effect of fixation temp and duration, and salt
conecntaration.
They also tried a large number of diverse
exratction tecniques
and describe the ne they found to be most
eefective.
-----------------------------------------------------
TI- MODIFICATIONS OF HUMAN AND
VIRAL DEOXYRIBONUCLEIC-ACID BY
FORMALDEHYDE
FIXATION
AU- KARLSEN, F;KALANTARI,
M;CHITEMERERE, M;JOHANSSON,
B;HAGMAR, B
NA- NORWEGIAN RADIUM HOSP,INST
CANC RES,DEPT PATHOL,N-0310
OSLO,NORWAY
HUDDINGE HOSP,DEPT CLIN VIROL,S-
14186 HUDDINGE,SWEDEN
JN- LABORATORY INVESTIGATION
PY- 1994
VO- 71
NO- 4
PG- 604-611
AB- BACKGROUND: Formaldehyde reacts
with human and viral DNA through
interaction with hydrogen bonds, fixation of
DNA-protein, and
hydroxymethylation of the nucleic acids. Even
though most archival
tumor tissues are fixed with formaldehyde,
little has been done to
analyze the consequences of the reaction of
formaldehyde with DNA,
Misleading results can be obtained from fixed
tissue using polymerase
chain reaction (PCR) typing or restriction
fragment length polymorphism
analyses.
EXPERIMENTAL DESIGN: We have
studied variations in fixation time in
various tissues obtained at autopsy and in
prostatic carcinoma biopsies
to analyze the effects of the formaldehyde
fixation. Different
PCR-products were studied after different
fixation times.
RESULTS: DNA from fixed tissues appears
to be no more fragmented than
the native DNA. Changes in the DNA
structure is more important than DNA
quantity for performing PCR on fixed tissues.
PCR products longer than
2 to 300 bp was difficult to amplify from
some tissues. Only 8 hours of
fixation can be enough to inhibit amplification
of more than 421 bp.
Tissue fixed for longer than 215 hours cannot
be amplified for more
than 200 basepair products unless excessive
numbers (50-80) of
PCR-cycles are used.
CONCLUSIONS: The loss of PCR product is
related to fixation time and
PCR-product-length, probably because of the
rate of denaturation
followed by modification of DNA, Contrary
to what has previously been
assumed, formaldehyde neither fragments nor
reduces the quantity of
DNA, but rather changes the structure of
DNA. Different tissues may
also react differently with formaldehyde, in
part because of different
tissue fixation gradients. When the PCR
product is shorter than 200 bp,
DNA isolated from paraffin-embedded tissues
fixed with 4% formaldehyde
can be useful to any kind of PCR product
analysis.
TI- THE EFFECT OF FORMALIN FIXATION
ON RESTRICTION-ENDONUCLEASE
DIGESTION
OF DNA AND PCR AMPLIFICATION
AU- HAMAZAKI, S;KOSHIBA,
M;HABUCHI, T;TAKAHASHI,
R;SUGIYAMA, T
NA- KYOTO UNIV,FAC MED,DEPT
PATHOL,SAKYO KU,KYOTO 606,JAPAN
KYOTO UNIV,FAC MED,DEPT GERIATR
MED,KYOTO 606,JAPAN
KYOTO UNIV,FAC MED,DEPT
UROL,KYOTO 606,JAPAN
JN- PATHOLOGY RESEARCH AND
PRACTICE
PY- 1993
VO- 189
NO- 5
PG- 553-557
AB- The effect of formalin fixation on DNA and
on polymerase chain reaction
(PCR) amplification was investigated. Lambda
phage DNA fixed in
buffered formalin showed incomplete
digestion on restriction
endonuclease treatment. The resistance to
restriction digestion was
dependent on the temperature of fixation, but
not affected by salt
concentration of the fixative.
Lambda phage DNA fixed in unbuffered
formalin showed poor PCR
amplification due to degradation of DNA
during fixation. Lambda phage
DNA fixed in buffered formalin evaded
degradation and suited for
template of amplification. Feasibility of
formalin-fixed tissues as
sources for PCR amplification was also
investigated with primers
producing 128 bp fragment of c-Ki-ras exon
2. Although DNA from tissues
fixed for 3 months showed amplification,
there was no amplification
from tissues kept in unbuffered formalin for
longer than 6 months.
TI- THE EFFECT OF FORMALIN FIXATION
ON DNA AND THE EXTRACTION OF
HIGH-MOLECULAR-WEIGHT DNA
FROM FIXED AND EMBEDDED TISSUES
AU- KOSHIBA, M;OGAWA, K;HAMAZAKI,
S;SUGIYAMA, T;OGAWA, O;KITAJIMA, T
NA- KYOTO UNIV,FAC MED,DEPT
PATHOL,YOSHIDA-KONOE-CHO,SAKYO
KU,KYOTO
606,JAPAN
KYOTO UNIV,FAC MED,DEPT GERIATR
MED,KYOTO 606,JAPAN
KYOTO UNIV,FAC MED,DEPT
UROL,KYOTO 606,JAPAN
KYOTO UNIV,FAC MED,DEPT
DERMATOL,KYOTO 606,JAPAN
JN- PATHOLOGY RESEARCH AND
PRACTICE
PY- 1993
VO- 189
NO- 1
PG- 66-72
AB- The effect of formalin fixation on DNA and
extraction of DNA from fixed
tissues was investigated to retrieve archival
tissue samples stored in
pathology departments for molecular
biological studies. Aldehyde
fixatives resulted in degradation of DNA at
room temperature but not at
4-degrees-C. The degradation also occurred in
formalin when the pH or
the salt concentration was low, or the formic
acid level was high.
Restriction endonuclease digestion of fixed
DNA was incomplete after
formalin fixation and this was also
temperature-dependent. Thus,
relatively intact DNA was obtained from the
tissues fixed in buffered
formalin at 4-degrees-C or fixed with
microwave irradiation. The use of
modified tissue-lysing buffer containing 4M
urea allowed extraction of
high-molecular-weight DNA suitable for
Southern blot analysis from
fixed and embedded tissues. In conclusion,
fixation with buffered
formalin at 4-degrees-C permitted extraction
of DNA of sufficient
quality for Southern blot analysis.
Good luck
Guy
Newcastle University
Dpt of Marine Sciences and Coastal
Management
