Chapter 5
Introducing Students to Conservation Genetics Using
Sturgeon Caviar
Kathleen A. Nolan, Tony Catalano, Phaedra Doukakis, Vadim
Birstein, and Rob DeSalle
Kathleen A. Nolan
St. Francis College
180 Remsen Street
Brooklyn, NY 11201
Anthony Catalano
St. Francis College
180 Remsen Street
Brooklyn, NY 11201
Phaedra Doukakis
Vadim Birstein
Rob DeSalle
American Museum of Natural History
Central Park West at 79th Street
New York, NY 10024
Kathleen A. Nolan is an Assistant Professor in Biology at St. Francis College,
Brooklyn, NY. She is a Visiting Scientist at the American Museum of Natural
History in the laboratory of Rob DeSalle. (knolan@worldnet.att.net)
Tony Catalano is a Biology student at St. Francis College who helped to develop
this lab exercise. He is interested in pursuing a career in forensic science.
Phaedra Doukakis is a Biology Ph.D. student at Yale University who laid the
groundwork for this lab exercise.
Vadim Birstein is the director of the Sturgeon Society, New York, NY.
Rob DeSalle is the curator of the Molecular Biology Lab in the Department of
Entomology at the American Museum of Natural History in New York, NY, and
a Professor at Yale University.
©2000 Kathleen Nolan/St. Francis College
Association for Biology Laboratory Education (ABLE) ~ http://www.zoo.utoronto.ca/able
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PCR of Caviar
Reprinted From: Nolan, K., T. Catalano, P. Doukakis, V. Birstein, and R. DeSalle. 2000. Introducing students to
conservation genetics using sturgeon caviar. Pages 102-112 in Tested studies for laboratory teaching, Volume 21 (S. J.
Karcher, Editor). Proceedings of the 21st Workshop/Conference of the Association for Biology Laboratory Education
(ABLE), 509 pages.
- Copyright policy: http://www.zoo.utoronto.ca/able/volumes/copyright.htm
Although the laboratory exercises in ABLE proceedings volumes have been tested and due consideration has been given to
safety, individuals performing these exercises must assume all responsibility for risk. The Association for Biology
Laboratory Education (ABLE) disclaims any liability with regards to safety in connection with the use of the exercises in its
proceedings volumes.
Contents
Introduction.............................................................................................................103
Materials .................................................................................................................104
Notes to Instructor...................................................................................................106
Student Outline .......................................................................................................107
Protocol: Week One................................................................................................107
DNA Isolation.........................................................................................................107
PCR Amplification..................................................................................................108
Electrophoresis........................................................................................................109
Photography ............................................................................................................111
Week Two...............................................................................................................111
Acknowledgements.................................................................................................112
Literature Cited .......................................................................................................112
Introduction
Most museums of natural history have as one of their missions uncovering the relatedness
among species. Traditionally, scientists have used measurements of physical characteristics,
embryology, and the fossil record to help determine this relatedness. Many scientists are now
using molecular biology tools in addition to these traditional approaches.
The Molecular Biology Laboratory in the Department of Entomology at the American
Museum of Natural History in New York City is interested in uncovering relatedness among
many different types of species (not just insects!). For example, molecular approaches have been
used to assist in the construction of a “family tree” (phylogeny) of 25 sturgeon and paddlefish
species (Order Acipenseriformes). These are the caviar-producing species.
The three species that produce the caviar that is most often found in U.S. delis (and now
over the Internet) are the beluga (Huso huso), the sevruga (Acipenser stellatus) and the osetra
(Acipenser gueldenstaedti). All sturgeon species are suffering from over-fishing and many are
trying to sruvive despite environmental degradation.
The land-locked Caspian Sea
commercially-fished species (the three mentined above) have perhaps received the most
publicity regarding their exploitation.
As caviar commands a high price in the marketplace, these fish are sometimes illegally
caught. All sturgeon species were placed on the Convention for Trade in Endangered Species
(CITES) list in 1998. What is worse, is that sometimes caviar from the more “commonly
available” sturgeon are replaced by even rarer species. The rationale behind being able to
identify “unknown” or “mistakenly labeled” caviar might lead to stricter laws against illegal
sales of caviar from endangered species in the future.
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When female sturgeon are caught, they are first stripped of their roe. The fish eggs are
packed carefully in salt to add to the flavor and so that they will keep for several months in an
unopened jar. After the salt is added, the roe is kneaded in a certain fashion---this is the “art” to
packing caviar (Cullen, 1999). When the jars are opened, the eggs should keep for several weeks
if tightly covered. (I have isolated DNA from caviar that has been several months old--if tightly
covered and refrigerated, the eggs appear to keep their shape and integrity, which is necessary to
isolate DNA from an intact egg.)
Mitochondrial DNA genes have been sequenced for each of these species, and primers
for amplifying these genes have been synthesized (Birstein and DeSalle 1998).
Experiments have been conducted in which single caviar eggs obtained from jars
purchased in Manhattan delis have been tested for the three commercially available species of
origin. It was ascertained through PCR analysis that a large percentage of these jars (23%) were
“mislabeled” (DeSalle & Birstein 1996). In other words, the jars contained either: (a.) Species
that should not have been fished for because they were endangered or (b.) Caviar that should
have been sold at a lower price. As one of my Russian students put it, “There is a lot of fake
caviar out there.” Species ID kits are being developed for use in conservation and wildlife
forensics. It is thought that, since eggs from endangered species have been detected, illegal
fishing of endangered species has occurred. The students who elect to participate in this
laboratory exercise could thus contribute to a database on the occurrence of this and, in effect, be
conducting forensic science.
Materials
Sturgeon caviar. Buy inexpensive black caviar from a food import deli.
Sevruga and osetra are the least expensive, and costs about $25 for a half ounce. Caviar is also
now being sold over the Internet. This should yield hundreds of eggs, which are a little
larger than the head of a pin.
Gloves
Quanta-GenomicTM Kit--QUKG-50 (this kit provides enough material for 50 samples): Quantum
Biotechnologies Inc., 1801 de Maisonneuve Blvd., West 8th floor, Montreal, Quebec,
Canada H#H 139, Tel: 888-DNA-KITS--514-935-2200---Fax: 888-688-3785--514-935-7541,
e-mail: info@gbi.com, Internet: http///www.quantumbiotech.com
100% ethanol
70% ethanol
RNAse free water --Amersham-Pharmacia Biotech--1-888-573-4732, cat. #US 70783 (contains
.01% diethylpyrocarbonate) $58 for 1 liter, $35 for 500 ml.
Distilled water
DNTP’s (make a master mix of 10mM each of the 4 dNTP’s), dNTP Kit - Amersham-Pharmacia
Biotech, cat. #27203201, $67.
PCR buffer (10X) (from Perkin Elmer to go with AmpliTaq. This buffer should contain 15 mM
magnesium chloride. Some types of PCR buffers are not supplemented with magnesium
chloride---in that case, it must be added separately.)
Primers are ordered going from the 5' to the 3' ends. In this exercise, we will use three
primers: B72 and S2A should amplify a 150 base pair fragment of a cytochrome b gene that has
been sequenced and found to be in all sturgeon. When S2 is used with S2A, only a cytochrome b
gene fragment (approximately the same size) specific to sevruga sturgeon (Acipenser stellatus)
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should be amplified. We ordered ours from Operon Technologies, 1-800-688-2248. There is
what is called a $5 set-up fee and the cost is 60 cents per base. The primers are reconstituted
upon arrival with RNase free water. These come dehydrated in pico to nanomoles quantities,
and should be diluted to 1-10mM. Each primer will vary slightly in concentration when it is
shipped to you. The primer is further diluted to nanomole quantities when it is used in the PCR
reaction mix.
Primer 1 (B72) GCCTACGCCATTCTCCG
Primer 2 (S2A) CCTCCAATTCATGTGACTACT
Primer 3 (S2) GGAGTCCTAGCCCTCCT
Taq polymerase (We use AmpliTaq from PE Biosystems, 1-800-327-3002, Cat. # N808-0160,
$155. This comes with the required PCR buffer (10X) supplemented with 15 mM
magnesium chloride. (Some types of PCR buffers are not supplemented with magnesium
chloride, in that case, it must be added separately.)
Agarose
2 X TBE buffer pH 8.0 (Tris-boric acid-EDTA buffer--can be purchased from Carolina
Biological, 1-800-334-5555, cat #219027, $25 for 500 ml, or made up yourself - see recipe
below - you would first make up a 10X solution, and then dilute to 1X as needed.)
To make one liter of 10X TBE fuffer:pH 8.0 add the following to 700 ml of distilled water in a
2-liter flask:
1 g of NaOH
108 g. of Tris base
55 g boric acid
7.4 g of EDTA
Stir to dissolve; bring to volume. (Micklos and Freyer, 1990)
NaOH (to adjust pH to 8.0 if necessary)
Hydrochloric acid (to adjust the pH)
To make 100 ml of 6X loading dye, dissolve:
0.25 g bromophenol blue
0.25 g xylene cyanol
in 49 ml of water. Stir in 50 ml of glycerol. (Micklos and Freyer, 1990)
0.025% methylene blue or
ethidium bromide (5 mg/ml) - HANDLE WITH CAUTION!! - (See Micklos and Fryer (1990) to
learn how to handle this mutagen.)
mineral oil
ice bath
minicentrifuge
micropipettors (1-20 µl and 1000 µl)
micropipettor tips (1-20 µl and 1000 µl)
(Some labs have micropettors that range from 0-10 µl; if this is the case, than an additional tip
size would be needed.)
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PCR of Caviar
Eppendorf tubes (0.5 ml and 1.5 ml)
Polymerase Chain Reaction (PCR) machine,
or
Water baths set at the appropriate temperatures for manual PCR (this is untested).
Gel electrophoresis set ups (trays, combs, gel chambers, power supplies)
Trays for staining with methylene blue and/or
Ethidium bromide
Plastic Rubber-maid type containers with covers in which to place gels for staining.
Photography equipment (optional)
(In this laboratory both Polaroid and a Kodak digital photography set-up in use with a
MacIntosh computer are used for photography). Life Technologies, 1-800-828-6686,
distributes the Kodak Digital Science EDAS 120. A Polaroid specifically for use with UV
can be purchased from Carolina Biological, cat. #213699, $450.
Light box--UV for viewing ethidium bromide stained gels and/or
White light for viewing methylene blue stained gels
Notes to Instructor
Level of difficulty: Upper undergraduate genetics course
Time required to prepare and set up: five to eight hours
Time required for students to perform exercise:
Two hours to isolate DNA and to make dilutions for the control primers; three hours for
the PCR reaction to run; one hour to run the gel.
A repeat of the above times is needed for the reactions with the species-specific primers.
I suggest that you take two weeks to complete this exercise; one week to do the control,
and the second week to do the species-specific reactions.
You will need to make a list of the total number of spots that are available on your PCR
machine(s). Each student (if working individually) will need three spots; however, I suggest that
one dilution (1:10) will probably work. Once you have the number of spots matched to the
number of students you have, make a similar list for the number of reactions, number of students,
and wells on a gel available based on type of comb and number of gel boxes.
The recipe for the reaction mix as given in the protocol is for one student. You need to
multiply these amounts by the number of students you have, plus extra for negative controls.
The number of negative controls will be determined by the number of wells you have on your
gel, and how many students are using each gel.
This experiment has been successfully tested with the species-specific Acipenser stellatus
primers. There are primers available for the Huso huso caviar, but this caviar is twice as
expensive as the sevruga, and therefore probably not practical for this exercise. Also, the osetra
primers will hybridize to species closely related to the Acipenser gueldenstaedti so they are
considered to be species-specific enough for this exercise. The most precise way to determine
whether a certain caviar egg is from a specific species is to sequence the DNA, which goes
beyond the scope of this exercise.
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Student Outline
Protocol--Week One
Isolation of DNA from single caviar eggs
Lysis and homogenization step
Note: The Quantum kit components come in four bottles labeled GEN I through GEN IV.
(These kits contain proprietary solvents, which the company representatives, when called
on the phone, would not reveal what they were. A good biological techniques book (Robyt
and White, 1987, is one example) should describe how traditional ingredients such as
detergents and ethanol work in lysing cells and precipitating DNA.)
1.
Add 300 µl of GEN I to each of two 1.5 ml microfuge tubes.
2.
Add one sturgeon egg from a microfuge tube marked “S” for sevruga to the first tube with
a yellow 20 µl pipette tip, and “smash the eggs open” against the side of the tube. You
should see white material oozing from the egg. In the second tube repeat the process with
an egg from a microfuge tube marked “O” for osetra.
3.
Add 300 µl of GEN II to each tube and mix by inverting the tube several times.
4.
.
5.
Incubate the tubes at 55o C for 30 minutes.
Add 150 µl of GEN III to each tube and mix vigorously by inverting the tubes twenty
times.
6.
Leave the tubes at room temperature for 10 minutes, mixing them occasionally by inverting
them.
7.
Centrifuge the tubes at high speed in a microfuge (14,000 RPM) for 5 minutes.
8.
Transfer the supernatants to fresh labeled tubes, and discard the tubes with the pellets.
9.
Add 450 µl of isopropanol to each tube of supernatant and mix well.
10.
Centrifuge the tubes at high speed (14,000 RPM) for 5 minutes. Discard the supernatants.
11.
Rinse the precipitate in each tube with 200 µl of 70% ethanol. Centrifuge the tubes for 5
minutes. Discard the supernatants.
12.
Allow the pellets to air dry until there are no drops of alcohol left--this step may be aided
with a hair dryer at a low setting.
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PCR of Caviar
13. Resuspend each pellet in 20 µl of RNAse-free water.
PCR Amplification
1.
You may warm the sample for a few minutes in a 55oC water bath, if the DNA appears to
not have gone into solution.
2.
Since you are not sure of the quantity of DNA that you have isolated, and the PCR reaction
is DNA concentration-dependent, it will be necessary for you to do a dilution series of your
DNA. (You need to see how many PCR machines you have, or how many spots you have
available to you on a PCR machine to see if it is practical for your class to do the
amplifications of every dilution. If you don’t have a lot of room on the machines, use a
1:10 dilution of the DNA for the PCR reaction.
3.
Set up two 1.5 ml tubes for the sevruga DNA and label them 1:10, 1:100.
process for the osetra DNA.
4
Place one µl of the appropriate DNA in the appropriately labeled tube. Add the RNAsefree water to make the dilution. (Check with your instructor first to make sure that you are
adding the correct amount of water.)
5.
To set up the PCR mix:
Repeat the
Add 1 µl of the appropriate dilution to a 0.5 ml (PCR) tube. You should have three PCR
tubes: one for the undiluted DNA, and the other two for the two dilutions that you made.
Next, add 24 µl of the PCR reaction mix that has been prepared by the instructor and is
sitting on ice.
The control reaction mix contains the following ratios of reactants:
19 µl RNAse free water
2.5 µl of PCR buffer supplemented with 15 mM magnesium chloride
2.5 µl of DNTP’s (10 mM each)
0.1 µl of primer 1 (B72)
0.1 µl of primer 2 (S2A)
0.1 µl of Taq polymerase
6.
Spin tubes briefly at top speed in the microfuge for a few seconds.
7.
Add two drops of mineral oil from a dropper bottle to each tube.
8.
Spin tubes briefly at top speed in the microfuge for a few seconds.
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PCR of Caviar
9.
Place the PCR tubes in the PCR machine. Run the PCR reaction under the following
parameters:
35 cycles of:
94o 1 minute
48o 1 minute
72o 1 minute
10.
When the samples are finished, place them at 4o C until you have time to complete the
electrophoresis.
Electrophoresis.
1.
Place 1 gram of agarose into a 125 ml Erlenmeyer flask. Add 50 ml of 1X TBE buffer.
2.
Microwave the solution until it boils (about a minute and a half). Alternatively, heat the
agarose in buffer to boiling on a hot plate. Make sure that all the agarose is dissolved, but
don’t burn it. (What percentage gel would this be?)
3.
Let the gel cool at room temperature till hot but not burning to the touch. (Touch the flask
to your cheek!)
4.
At this point, add 0.5 µl of a 5 mg/ml solution of ethidium bromide to the agarose.
CAUTION!! Since ethidium bromide intercalates between the bases of DNA, it is a
potential carcinogen. Handle with care. Omit this step if you are using the methylene blue
method of DNA staining.
5.
Cast the gel according to the directions that come with your gel tray--you can use tape to
seal the ends, or some trays come with rubber “gaskets” that fit over the ends and
effectively seal it. Place your tray on a level surface. Place a 8-12 well comb into the tray.
Pour the gel quickly. Push any bubbles to the side with a yellow micropipet tip.
6.
The gel should be hardened after about 20 minutes--it will turn opaque and you may see
wavy lines running through it.
7.
Pull out the comb by wiggling it gently as you pull--this takes a little practice. Take off the
tape or the rubber gaskets.
8.
Place the gel in an electrophoresis chamber. Cover the gel with 1X TBE. You should just
barely cover the gel-- the less buffer, the less resistance to the current. As you increase the
amount of buffer, you will notice that by switching to the "current" or "ampere" setting, the
number of amperes increases, which is undesirable because this could create too much heat
and cause your gel to melt.
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PCR of Caviar
9.
If necessary, you may practice pre-loading the gel with loading dye, and then run the dye a
few centimeters through the gel by turning on the voltage to 125 V.
10.
Prepare to load the gel. Take a piece of parafilm around 10 cm square and place it down
flat on the lab bench.
11.
Add 2 µl of dye as dots spaced apart on the parafilm--one for each sample that you will
load.
12.
Spin tubes briefly at top speed in the microfuge for a few seconds and take off as much
mineral oil as possible with a micropipette. You must do this very slowly and carefully in
order to avoid taking out your sample along with the oil.
13.
Make a key of the order of your samples that you will be loading into your gel in your
notebook.
14.
Mix your samples with loading dye, which: (a) contains glycerol which weighs the
samples so that they do not float out of the gel and (b) contains two dyes that will separate
upon migration through the gel. Add 8-10 µl of each sample to the blue loading dye dot
and mix by pipetting up and down once. Take the sample from the bottom of the tube so
that you won’t get any mineral oil in your sample. (The mineral oil won’t mix with the
dye.)
15.
Carefully add your samples to the wells. Turn on the power supply and set the voltage at
125 volts. You may go up to 150 volts, but heating the gel for too long a period of time
may distort it.
16.
You will notice that the loading dye will separate out into a dark blue and a light blue band.
The faster-moving dark blue band will co-migrate with a DNA fragment that is
approximately 300 base pairs in length, and the slower-moving light blue band will comigrate with a DNA fragment that is approximately 9,000 base-pairs in length.
17.
Turn off the power supply, remove the gel tray, and carefully slide the gel into a staining
chamber.
18.
If you used ethidium bromide, cover your gel with distilled water. If you want to use
methylene blue for staining, cover the gel with 0.025 % methylene blue.
19.
The ethidium bromide gel may be ready for photography. View the gel on the UV light
box. Be sure to use a protective face shield or box cover, and determine if bright clear
bands are present. If the bands are not differentiated enough from the background, destain
the gel by leaving it in the distilled water for an hour to several hours.
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PCR of Caviar
20.
If you used methylene blue, leave the gel in this solution for 30 minutes. Destain the gel by
pouring off the methylene blue solution into a storage container, and running the gel under
tap water for a few minutes. Leave the gel in tap water for several hours; the bands will
become more visible with destaining.
Photography
A. Digital photography
The laboratory at the American Museum of Natural History is equipped with a Kodak camera
that takes digital pictures and captures them to a computer. A more traditional set-up is the
Polaroid camera. We think that every lab should aim toward a digital set-up, and try to obtain
grant money for this.
If you have a digital camera, follow the directions for taking a picture. If you save the image
as a TIF or PIC file, it will use up less room on your disc than the Kodak graphics save. Then
you may print a copy of your photo, using the Adobe graphics program.
B. Polaroid photography
For UV photography of ethidium bromide stained gels, use Polaroid high-speed film Type
667 (ASA 3000). Set the camera aperture to f/8 and shutter speed to B. Depress shutter for a 2-3
second time exposure.
For white light photography of methylene blue stained gels, use Polariod Type 667 film,
with an aperture of f/8 and a shutter speed of 1/125 second. (You may have to play with these
settings!)
Results
You should see nothing in your negative control lane and a nice, 150 base-pair band in
your sample lanes. If you get this result, then you have successfully amplified and visualized a
150 bp fragment of a sturgeon cytochrome b mitochondrial gene!
This is your control; you will now repeat the species-specific experiment substituting the
S2 primer for the B7-2 primer.
Week Two. Species-specific DNA PCR amplification
Take your DNAs from both the osetra and the sevruga caviar and test it with the sevruga
species specific primers (S2 and S2A). For these PCR amplifications use the dilution that gave
you the brightest band for the control primers. This set of primers should give a positive
amplification for any egg that is from the species Acipenser stellatus, or the sevruga caviar.
Repeat from Step 5 under “PCR Amplification, substituting one tube for the three tubes with the
three different dilutions. Follow the protocol through the “Eletrophoresis” and “Photography”
sections. Note that the primers and the PCR reaction conditions are different. Note your
results.
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PCR of Caviar
The experimental or species-specific reaction mix has been prepared to contain the
following ratios of reactants:
19 µl RNAse free water
2.5 µl of PCR buffer supplemented with 15 mM magnesium chloride
2.5 µl of DNTP’s (10 mM each)
0.1 µl of primer 1 (S2) Note that this primer is different from that in the control.
0.1 µl of primer 2 (S2A)
0.1 µl of Taq polymerase
The PCR reaction conditions are slightly different for the sevruga species-specific reactions:
35 cycles of:
94o 1 minute
55o 1 minute
72o 1 minute
If the species is not Acipenser stellatus (or sevruga), there should be no bands that will
light up. Make sure that you set up your gel so that you have one lane that contains some
positive control DNA (from Week One).
Primers that are sturgeon species-specific are still being sought in order to differentiate
endangered from non-endangered sturgeon species. You have just taken a step closer to
becoming a wildlife forensic scientist!
Acknowledgements
This project was funded, in part, by a Faculty Research Grant from St. Francis College and by a
Laboratory Teaching Initiative Grant from the Association of Biology Laboratory Education.
Literature Cited
Birstein, V., Doukakis, P., Sorkin, B., and R. DeSalle. 1998. Population aggregation analysis of
three caviar-producing species of sturgeons and implications for the species identification
of black caviar. Conservation Biology 12:766-776.
Cullen, Robert. 1999. The rise and fall of the Caspian Sea. National Geographic 195:2-35.
DeSalle, R. and V. Birstein. 1996. PCR identification of black caviar. Nature 381: 197-8
Micklos, D. A. and G. A. Freyer. 1990. DNA Science: A First Course in Recombinant DNA
Technology. Cold Spring Harbor Laboratory Press and Carolina Biological Supply Co.
[ISBN 0-89278-411-3]
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