Notes on DNA from Formalin-fixed specimens

Guy Reeves R.Guy.Reeves at ncl.ac.uk
Thu May 18 07:54:48 EST 1995


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unpublished protocols for  600bp+ but 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?
-----------------------------------

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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









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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

Path: news.ncl.ac.uk!nntphost.dur.ac.uk!strath-
cs!str-ccsun!zippy.dct.ac.uk!uknet!daresbury!not-
for-mail
Newsgroups: bionet.molbio.evolution
Subject: re DNA from formalin-fixed specimen
Message-ID: <3oo93k$257 at mserv1.dl.ac.uk>
From: <monnerot at cgmvax.cgm.cnrs-gif.fr>
Date: 9 May 1995 18:34:44 +0100
Sender: lpddist at mserv1.dl.ac.uk
Distribution: bionet
Original-To: mol-evol at dl.ac.uk
Lines: 16

Anne-Marie Vachot, a student in my group, has 
been working on this problem 
for several months. She is able with a new 
protocol she has designed 
to extract DNA from formalin-fixed specimen 
and amplify 
succefully and repeatedely DNA fragments up to 
400 bp and sometimes 
to 600 bp. She has shown that sequences of PCR 
products are identical to 
that from fresh material.She will present her 
work at the ancient DNA 
meeting (Oxford july 20-22) and publish very 
soon a review from the literature
 on the effects of formalin, the new 
technique for extraction as well as new 
conditions for liquid preservation. 
Those interested on this subject, please contact 
me:
Dr Monique Monnerot
CGM, CNRS
F - 91198 Gif sur Yvette Cedex
Tel 33 1 69 82 43 58
FAX 33 1 69 07 49 73
E-mail Monnerot at cgmvax.cgm.cnrs-gif.fr

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




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