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Hi All,
Well the replies have nearly stopped coming in to my initial question of
obtaining DNA from tissues that were stored in formalin. Thanks to all
those who replied. Judging from the number of requests I received to
forward on any information from this question it is obvious that tissues
stored in formalin are an important hinderance to molecular biologists.
There are many techniques available (most of them slight variants), but it
seems the most crucial step in soaking of the tissue for a few days to
remove any excess formalin that may be present. Here are all the
references to papers and protocols that I was sent. If anybody has any
further ideas or queries please drop me a note. Good luck everybody and
please other fisheries biologists, unless there is a specific need to
preserve specimens in formalin, just use alcohol so the specimens are easily
amenable to molecular work,
Cheers,
Dean
Protocol 1.
To extract DNA, whole individuals were placed in
microfuge tubes with 200ul of tissue lysis buffer ATL, from
the QIAamp, tissue extraction kit (Qiagen, Chatsworth, CA,
USA) and incubated for 24 hr at 55!C. Then 5ul of a 50mg ml-
1 solution of Proteinase K and an additional 95ul of lysis
buffer were added and incubation continued at 55!C for an
additional 72 hr (Goelz et al. 1985). The extraction then
followed the manufacturers instructions, except that buffer
AL and ethanol were increased from 200ul to 300ul. DNA was
eluted with one 200ul aliquot of 10mM Tris pH 8.0.
Because fresh deep-sea specimens were not available
for primer development, we targeted 16S rDNA by designing
primers to conserved regions within shallow-water
protobranchs. A forward primer (5U-AWR WGA CRA GAA GAC CCT-
3U, Proto16F) internal to 16sar (Kocher et al. 1989; Palumbi
et al. 1991) was designed by aligning two protobranch
bivalve species, Nucula proxima and Solemya velum. A
reverse primer 16R3, 5U-GCT GTT ATC CCT RNR GTA ACT-3U,
internal to 16sbr (Kocher et al. 1989; Palumbi et al. 1991)
was designed by aligning N. proxima, S. velum and Homo
sapiens. These primers (Proto16F and 16R3 (anneal
temperature 50!C)) were used to initially amplify DNA
isolated from preserved deep-sea specimens of Deminucula
atacellana.
These primers were degenerate, required low annealing
temperatures, and only worked sporadically. To improve
amplification success, we developed species-specific primers
for Deminucula atacellana by amplifying the whole 16sar and
16sbr fragment (557 bp) in two pieces. A primer internal to
Proto16F and 16R3 was designed, Proto16R 5U-CYC YCA GTT GCC
CCA ACT MAA-3U from the DNA sequence of the 16S fragment
amplified from D. atacellana. Template DNA from D.
atacellana was amplified with Proto16R and 16sar (anneal
temperature 55!C), resulting in a 326 bp fragment. The
fragments were sequenced, aligned and used to re-design a
species-specific primer, Demi16F 5U- GAC GAG AAG ACC CTA TTG
AGT -3U in the same area as Proto16F but extending it
several bases at the 3' end to avoid similarity to human
DNA. Template DNA from D. atacellana was then amplified
with Demi16F and 16sbr (anneal temperature 60!C) and the 330
bp fragment sequenced. Sequences were aligned and used to
re-design a primer Demi16R, 5U-GAT TAC GCT GTT ATC CCT RTG-
3U, conserved to N. proxima and D. atacellana in the same
area as 16R3.
Developing the species-specific internal primers and
working with small (<300bp) mtDNA fragments were crucial
steps in successfully working with formalin-fixed
individuals. The species-specific primers Demi16F and
Demi16R were used to amplify a 196 bp fragment of DNA from
89 individuals. Individuals were amplified in 50ul reaction
volumes consisting of 10ul template (no dilution of stock
DNA eluted from column), 50mM KCl, 10mM Tris-HCl (pH 9.0),
0.1% Triton X-100, 2.5mM MgCl2, 0.2uM each dNTP, 20pm each
primer, 1.0 unit Taq (Promega, Madison, WI, USA), an equal
volume of Taqstart Antibody (Clonetech, Palo Alto, CA, USA)
and H20 to 50ul. Reactions were layered with mineral oil and
heated to 95!C for 2 minutes prior to 5 cycles of 95!C for 1
minute, 60!C for 1 minute and 72!C for 1 minute, then 35-40
cycles of 94!C for 30 sec, 60!C for 30 seconds and 72!C for
30 seconds. After amplification we confirmed and purified
PCR products by running them out on 1.5% agarose gels and
purifying with QIAquick PCR purification kit (Qiagen,
Chatsworth, CA, USA). PCR products were sequenced with a
Taq Dye Deoxy Termination cycle sequencing kit (PE Applied
Biosystems, Foster City, CA, USA), ethanol precipitated and
run on an Applied Biosystems Model 373 Automated DNA
sequencer.
Protocol 2.
Dean,
A fisheries consultant we work with in Ft Collin, Colorado, Bill Manci,
passed your message about PCR from fish tissue samples on to me. We do
do a lot of DNA preps and subsequent PCR on fish tissue samples; in our
case primarily looking for the presence of infectious disease
organisms. So I have a number of thoughts:
(1) Preserve the samples in 70% ethanol instead of formalin if possible.
(2) Wash formalin preserved samples in 70% ethanol before proceeding
(3) Use a spin column DNA prep procedure, like those sold by Qiagen,
instead of phenol-chloroform procedures.
(4) To eliminate any question about the quality of DNA (for PCR) from a
formalin preserved sample, make a dilution series of the DNA prep spiked
with known positive amplifiable DNA.
We will be receiving some exclusively formalin preserved samples in the
next month or so and will run a spiked dilution series on these samples
to confirm limited data that we already have that spin column DNA preps
are unaffected by formalin.
Protocol 3
>1. use a small piece of tissue (ca 0.5cm square)
>2. wash the tissue for 3 successive 24hour periods (rotary shaker on low
>rpm, room temp) in fresh solutions of 10ml of 1xGTE (100mM Glycine, 10mM
>Tris-HCl pH8, 1mM EDTA). This acts as a binding agent for excess formalin.
>3. Air dry the tissue and add 500microlitres of extraction buffer (1percent
>SDS, 25mM TrisHCl ph 7.5, 100mM EDTA).
>4. Add 20microlitres of 1M DTT and 100microlitres of 10mg/ml Prot K
>5. Digest at 65C for 24h.
>6. Add an additional 25microlitres of prot. k (20mg/ml, dont ask me why its
>a different concentratyion from the first lot..probably doesnt make any
>difference). and 10microlitres of DNase-free RNase (10mg/ml, dont ask me
>why people use this. RNA will be completely degraded after so many years
>and I cant see that the presence of RNA would affect PCR anyway. Just
>another source of contamination if you ask me!) after the first 10h of
>digestion.
>7. Then do 3x phenol extractions (500microlitres per time. If you can
>afford it, buy in pre-equilibrated phenol....it saves time, effort, reduces
>contamination risk)
>8. Extract 2x with phenol:chloroform:IAA (25:24:1), 500microlitres per go.
>9. Extract twice with chloroform:IAA
>10. Precipitate DNA by ading 2.5 vol cold (-80C) abs EtOH and place
>immeiately in the freezer (-20C) for 24h. (I use Microcon-100 instead of
>EtOH pptation. I can tell you about it if you want)
>11. Spin DNA for 30 min at 10000xg
>12. Remove EtOH and rinse pellet 2x with cold 70 percent EtOH, and then
>thoroughly air dry the pellet.
>13. Resuspend the pellet in 40microlitres of 1xTE (pH8).
References
Shiozawa et al. 1992. DNA extraction from preserved trout tissues.
Great Basin Naturalist 52(1):29-34.
BioTechniques, 22(3):394-400.
Acta Neuropathologica 93(4):408-413
Acta Neuropathologica 88(1):19-25
Diagnostic Molecular Pathology 5(3):220-224
Histochemical Journal 26(4):377-346.
Histology and Histopathy 12(3):595-601
Insect Molecular Biology 5(1):21-24
International Journal of Oncology 5(3):453-457
Journal Forensic Sci 42(6):1032-1038
Neoplasma 43(2):75-81
Pathology International 46(12):997-1004
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.
Dean Jerry
Animal Conservation Genetics
School of Resource Science and Management
Southern Cross University
Lismore
NSW 2480
Australia
Ph: 61 66 203815
Fax: 61 66 212669
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If we possessed a thorough knowledge of all parts of the seed of any
animal, we could from that alone, by reasons entirely mathematical and
certain, deduce the whole conformation and figure of each of its members,
and, conversely if we knew several peculiarities of this conformation, we
would from those deduce the nature of its seed.
Rene` Descartes 1596-1650 (Oeuvres iv, 494)
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