Solving a Crime Using DNA
Analysis and Chemistry
(basic forensics) Student
Manual
I.
Purpose and Background ....................................................................................1
II.
Protocol for Crime Scene Analysis .....................................................................3
III.
I.
A.
Materials Required ..........................................................................................3
B.
The Scenario ...................................................................................................3
C.
Chromatography of Marker Inks ......................................................................4
D.
RFLP Analysis of DNA Samples .....................................................................5
!
This student manual is
available online only.
Reproduction for noncommercial educational purposes only. Copyright
2008, 2010 Promega
Corporation. All rights
reserved.
STR-Based Analysis of DNA Using Silver Stain Gel .........................................8
A.
Before You Begin .............................................................................................8
B.
Amplification ....................................................................................................9
C.
Polyacrylamide Gel Preparation ....................................................................11
D.
Polyacrylamide Gel Electrophoresis ..............................................................14
E.
Silver Staining................................................................................................16
F.
Generating Film Images ................................................................................17
Purpose and Background
Forensic scientists and geneticists often talk about “DNA Fingerprinting” as a major
technique in their work. DNA Fingerprinting uses several techniques including: 1)
DNA purification, 2)amplification of DNA fragments, 3) separation of fragments, and
4) detection of differently sized fragments revealing the DNA “fingerprint”. These
result in a representation of genetic differences between individuals. The two most
commonly used methods are Restriction Fragment Length Polymorphism (RFLP)
analysis and Short Tandem Repeat (STR) analysis.
Several technological advances have contributed to the development of modern
DNA fingerprinting analysis.These include work in the late 1970s with restriction
enzymes to show that DNA could be cut into discrete and predictable fragments
based on the sequence of bases in the DNA molecule. Changes in the DNA
sequence could affect the sites where the restriction enzymes cut the DNA and
change the fragments produced from a restriction digest.
Once a DNA sample has been subjected to restriction enzyme digestion, the
digested fragments can be separated by gel electrophoresis. The DNA is loaded into
an agarose or polyacrylamide gel. The rate that the DNA migrates through the gel
matrix is determined by the size of the fragments. Since DNA is negatively charged,
if the gel is placed in an electrical gradient, the DNA fragments will be attracted to
the positively charged pole and “migrate” toward it. Scientists can digest the same
region of DNA from two samples and compare the way the fragments migrate. If
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there are changes in the banding pattern between the two restricted samples, then
there is probably a corresponding change in the sequence of the DNA that is
responsible.
The changes produced in the DNA restriction fragments as a result of changes in
the DNA sequence are known as Restriction Fragment Length Polymorphisms
(RFLPs).
In the early 1980s, Dr. Alec Jeffreys, a British geneticist, used RFLP analysis to
show that human genomic DNA contains repeated sequences and that these repetitive regions could be used for human identification because the number of times
these sequences are repeated one after another (in tandem) along a DNA molecule
varies from individual to individual. These repetitive regions, also known as Variable
Number of Tandem Repeat (VNTR) loci, are found throughout the genome. Further
more, the number of repeats is passed from parent to offspring, making VNTR
analysis useful for paternity determination.
Short tandem repeats (STRs) are a subset of VNTRs that are used in modern forensic analysis. STRs are located in regions of the genome that have no known function. The sequences are repeated head-to-tail at a specific point on a chromosome.
STR analysis involves PCR amplification and determination of the number of
repeats by looking at the sizes of the amplified fragments.
If you know of a restriction enzyme cut site on either side of the area of repeated
sequences, you can use restriction enzyme digestion to cut the repeated region
from the DNA molecule and compare the fragment sizes from different individuals.
Or, if you know the nonrepeating DNA sequences on either side of the VNTR, you
can design primers and use PCR to amplify the repeat and compare the size of the
amplified products from two individuals.
Scientists developed a technique for amplifying small amounts of DNA using DNA
polymerase in vitro. This technique called, Polymerase Chain Reaction (PCR),
allowed scientists to take a very small sample of DNA and make many copies of it
so that the DNA could be analyzed. PCR technology is part of any kind of crime
scene forensic analysis or genetic diagnosis that is based on a small sample.
The number of repeats at a single VNTR locus will not allow you to distinguish one
person from the rest of the population, so forensic scientists and geneticists look at
the fragment sizes generated from several different loci. The COmbined DNA Index
System (CODIS) is a federally maintained database that is used by law enforcement
in the United States. The database consists of 13 STR loci and a sex identification
marker, all of which are analyzed in forensic investigations. Because each locus provides two pieces of information about an individual (one for each chromosome),the
number of sites examined provides a greater power of exclusion to identify an individual based on a genetic profile.
In the first laboratory protocol, you will compare DNA from a crime scene with DNA
from two suspects using RFLP analysis.
If your instructor uses the second protocol, you will perform STR analysis at three
loci and determine gender based on the Amelogenin locus.
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II.
Protocol for Crime Scene Analysis (Chromatography and RFLP Analysis)
II.A. Materials Required
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
two black markers of different brands
500 ml beakers
acetone
XmnI restriction enzyme (5 units or 0.5 µl of 12 u/µl concentration)
HincII restriction enzyme (6 units or 1.5 µl of 10 u/µl concentration)
nuclease-free water
Buffer B (restriction enzyme buffer)
DNA sample from crime scene
DNA sample from suspect one
DNA sample from suspect two
Agarose LE, Analytical Grade (Cat.# V3121)
TBE Buffer, 10X (Cat.# V4251)
Ethidium bromide stock solution, 10 mg/ml
DNA gel electrophoresis apparatus and power supply
UV light box and camera or scanner
Pipettors and pipet tips
1.5 ml sterile tubes
DNA Markers (BenchTop pGEM® DNA Markers Cat.# G7521)
II.B. The Scenario: The Last Bag of Chocolate Chips
The manager of the college food services promised the president of the college a
plate of his favorite chocolate chip cookies for the upcoming meeting with the executive council of the Board of Directors. The chocolate chip cookies are a specialty of
Chef Lombardo's and are famous across the college campus. Almost like Pavlov's
dogs, students, professors, faculty and staff emerge from their offices and dorm
rooms in a grand migration toward the Dining Hall when the aroma of these cookies
backing drifts across campus. The appeal of these cookies can be traced, in part, to
the chocolate chips that are imported from Europe.
The day before the board meeting, Chef Lombardo checked the pantries in the main
kitchen and discovered that he only had one bag of these special chocolate chips
left. He placed all of the ingredients onto the lower shelf of the pantry so that he
would be ready to bake the cookies first thing the next morning. Chef Lombardo
locked the pantry, turned off the kitchen lights and left for the night.
When Chef Lombardo returned the next morning, he was greeted with a horrific
sight. The door to the kitchen had been pried open, and the metal door on the pantry
was bent and torn where someone had pried the lock of the pantry door. The ingredients for the cookies were scattered around the kitchen. The bags of sugar and
flour were busted on the floor. The open and empty bag of chocolate chips lay on the
kitchen counter along with a glass that had lipstick marks on it and a note printed in
black marker that read:
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This is just to say
I have eaten
the chocolate
that was in
the pantry
and which
you were probably
saving
for tomorrow.
Forgive me
it was delicious
so sweet
and so warm.
Apparently the thief had a literary bent, having adapted text from William Carlos
Williams for the thank you note. The thief had cut his or her hand on the pantry door
and left blood stains.
Chef Lombardo immediately called the president to report the tragedy. The criminal
justice, genetics and general chemistry professors were all then called to the crime
scene.
The investigators interviewed a group of students who had returned home late from
an outing and said that they had seen two professors walking back to campus at
2:00 am. The professors were walking toward the dining hall. One of the professors,
Dr. Johnson in history, sat on the executive council as the faculty representative to
the board and would have known about the chocolate chip cookies for the meeting.
Interestingly, both Dr. Johnson and the other professor, Dr. Lundquist in English
Language Arts, each had a hand bandaged when they were interviewed. The chemistry professor confiscated the black markers that were on each professor's desk for
further analysis. Each professor also gave buccal swab sample for DNA analysis.
II.C. Chromatography of Marker Inks
Lab Protocol
Note: Be sure to wear gloves when you handle the thank you note and your
own filter paper.
1.
Obtain a cut strip from the thank you note found at the
crime scene.
2.
Cut a clean piece of filter paper into two strips. Draw a
line across the width of the first strip using one of your
sample markers. Do the same thing with the other strip
and the other marker. Your mark should be about 2.5
cm above the bottom of the strip.
3.
Make a hole using a hole punch at the top of your evidence and sample
strips.
4.
Space all three strips along a glass stirring rod.
5.
Carefully add acetone to the beaker so that it will just cover the bottom
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0.5cm of the filter paper strips. The exact volume will depend on how far
down the strips extend into the beaker when they are suspended by the
glass stirring rod.
6.
Allow the acetone to wick up the filter paper strips. Once the acetone has
reached the top of the strips, remove them from the beaker and allow them
to dry. (Place them on a nonabsorbent surface or hang them to dry).
Analysis
1.
Can you see a difference between the inks of the two sample markers?
2.
Does either one of them clearly give the same chromatography pattern as
the ink on the note?
3.
Can you determine which type of marker was used to write the note? Why or
why not? If you think additional experiments are needed, what would you do?
II.D. RFLP Analysis of DNA
Before the Lab
Label 9 tubes for the following restriction enzyme digests:
Reaction
Crime scene sample Hinc II digest
Crime scene sample Xmn I digest
Crime scene sample HincII/XmnI digest
Suspect 1 sample Hinc II digest
Suspect 1 sample XmnI digest
Suspect 1 sample Hinc II/XmnI digest
Suspect 2 sample Hinc II digest
Suspect 2 sample XmnI digest
Suspect 2 sample Hinc II/XmnI digest
Tube #
1
2
3
4
5
6
7
8
9
Restriction Enzyme Digestion of DNA Samples
1.
Prepare the following master mixes for your single-enzyme digests as
directed in the table below. Prepare enough master mix for 8 digests to compensate for pipetting errors.
Master Mix for Single Digests
Component
Volume Needed for Each
Reaction
Volume Needed for 8
Reactions
Nuclease-Free Water
16.3µl
130.4µl
10X Buffer B
2.0µl
16.0µl
Acetylated BSA
0.2µl
1.6µl
2.
To the three HincII digests (Tubes 1, 4 and 7), add the following:
Single-Digest Master Mix
DNA (1µg/ml)
Hinc II (12 U/µl)
3.
18.5 µl
1.0 µl
0.5 µl
To the three XmnI digests (Tubes 2, 5 and 8), add the following:
Single-Digest Master Mix
DNA (1 µg/ml)
Xmn I (10 U/µl)
18.5 µl
1.0 µl
0.5 µl
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4.
Prepare the master mix for your double-enzyme digests according to the
table below. Prepare enough Master Mix for 5 digests to compensate for
pipetting errors.
Master Mix for Double Digests
Component
Volume Needed for Each
Reaction
Volume Needed for 5
Reactions
Nuclease-Free Water
15.8
79.0µl
10X Buffer B
2.0µl
10.0µl
Acetylated BSA
0.2µl
0.8µl
5.
To the three double-enzyme digests (Tubes 3, 6 and 9), add the following:
Double-Digest Master Mix
DNA (1µg/ml)
Hinc II (12 u/µl)
Xmn I (10 u/µl)
18.0 µl
1.0 µl
0.5 µl
0.5 µl
6.
Incubate the digests for 2 hours at 37°C.
7.
Heat inactivate the reactions by placing the tubes at 65 °C for 15 minutes.
Notes:
The double digest will result in 5 fragments for pGL4.12 and 4 fragments for
pGL4.11. This is because Xmn I does not cut pGL4.11.
Fragments from double digest of pGL4.12: 2215bp, 1306bp, 463bp, 328bp,
and 110bp.
Fragments from double digest of pGL4.11: 1306bp, 740bp, 110bp and
2214bp.
Agarose Gel Electrophoresis of Restricted DNA
1.
Prepare 1X TBE for your Gel Running Buffer.
2.
Weigh out the required amount of agarose, and add it to the appropriate
amount of 1X TBE buffer in a flask or bottle. For example to prepare a 2%
agarose gel, add 2.0 g of agarose to 100 ml of buffer.
3.
Heat the mixture in a microwave oven or on a hot plate for the minimum
time required to allow all of the agarose to dissolve. Interrupt the heating at
regular intervals, and swirl the contents. Do not allow the solution to boil
over.
3.
Cool the solution to 50–60 °C and pour the gel. Be sure to insert a gel comb
to create sample wells. Allow the gel to cool completely. Remove the comb
from the gel and place the gel in the electrophoresis apparatus.
4.
To analyze samples on the gel, prepare the following:
Enzyme digest
Blue/Orange Loading Dye, 6X
5 µl
1 µl
5.
Add enough 1XTBE gel running buffer to cover the gel.
6.
Load the samples onto the gel, and run at the voltage recommended by the
the gel box manufacturer.
7.
Run the gel until the orange dye front (runs at approximately the same rate
as a 50 bp piece of DNA) is near the bottom of the gel.
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8.
Remove the gel, and stain it by soaking it in a solution of 0.5 µg/ml ethidium
bromide (this is diluted from the 10 mg/ml) for 30 minutes at room temperature.
Note: Ethidium bromide is a carcinogen. Wear gloves and, be sure to dispose of it in accordance with your institution’s guidelines.
9.
Place the gel on a UV light box, and photograph the gel. Wear protective
eyewear when using the UV light box. If the gel is too orange, you can
destain the gel in water for a few minutes at room temperature.
Analysis
1.
Do either of your suspect DNA samples have the same RFLP pattern as the
crime scene sample?
2.
The two DNA samples probably have different restriction fragments. Can
you figure out why (what is the polymorphism that you were able to detect)?
3.
What is the purpose of the heat inactivation step at the end of the reaction ?
4.
How does agarose gel electrophoresis separate DNA fragments?
5.
TBE buffer is composed of tris, borate and EDTA. What function does each
of these ingredients serve in the buffer?
6.
What is the function of the running buffer during agarose gel electrophoresis? Can you use deionized water instead?
7.
Many chemicals stain DNA. Compare the way the following DNA stains
work: ethidium bromide, propidium iodide, methylene blue and DAPI.
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III. STR-Based Analysis of DNA and Silver Stain Gel
III.A. Before You Begin
This protocol will require running a polyacrylamide gel. You will need glass plates
and the accompanying gel electrophoresis apparatus. You can purchase precast 6%
polyacrylamide gels from GE Life Sciences and Invitrogen, but you will also need the
appropriate gel electrophoresis apparatus for the precast gels.
The quality of the purified DNA sample and choice of thermal cycler, as well as
small changes in buffers, ionic strength, primer concentrations, and thermal cycling
conditions, can affect amplification. We suggest strict adherence to recommended
procedures for amplification, denaturing gel electrophoresis, silver stain analysis and
recording data on film.
PCR-based STR analysis is subject to contamination by very small amounts of
human DNA. Extreme care should be taken to avoid cross-contamination when
preparing sample DNA, handling primer pairs, setting up amplification reactions and
analyzing amplification products. Reagents and materials used prior to amplification
(STR 10X Buffer, K562 Control DNA and 10X Primer Pairs) are provided in a separate box and should be stored separately from those used following amplification
(allelic ladders, STR 2X Loading Solution and pGEM® DNA Markers).
Always include a negative control reaction (i.e., no template) to detect reagent contamination. We highly recommend the use of gloves and aerosol-resistant pipette
tips.
Some of the reagents used in the analysis of STR products are potentially hazardous and should be handled accordingly. Table 1 describes the potential hazards
associated with such reagents.
Table 1. Hazardous Reagents
Reagent
Hazard
acetic acid (fix/stop solution)
acetic acid (fix/stop solution)
acrylamide
suspected carcinogen, neurotoxin
ammonium persulfate
oxidizer, corrosive
bisacrylamide
toxic, irritant
formaldehyde (staining solution and
developer solution)
highly toxic, suspected carcinogen
formamide (STR 2X Loading Solution)
irritant, teratogen
methacryloxypropyltrimethoxysilane
(bind silane)
toxic, moisture sensitive
silver nitrate (staining solution)
highly toxic, oxidizer
sodium thiosulfate (developer solution)
irritant, hygroscopic
TEMED
corrosive, flammable
urea
xylene cyanol FF (STR 2X Loading
Solution)
irritant
irritant
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III.B. Amplification
The GenePrint® STR Systems were developed for amplification without artifacts
usingTaq DNA polymerase. Use the buffer provided in the kit for your amplifications
with Taq DNA polymerase.
This protocol is for the amplification of CTT and amelogenin, allowing your students to
profile three STR loci and determine whether the DNA is of a male or female origin.
Materials to Be Supplied by the User
• GenePrint ® System (CSF1PO, TPOX, TH01; Cat.# DC6001
• GenePrint ® Sex Identification, Amelogenin (Silver Detection; Cat.# DC4081)
• Silver SEQUENCE™ Staining Reagents (Cat.# Q4132)
• thermal cycler, model 480 or GeneAmp® system 9600 (Perkin-Elmer)
• microcentrifuge
• Taq DNA polymerase (GoTaq® DNA Polymerase Cat.# M3001)
• Nuclease-Free Water (Cat.# P1193 or equivalent)
• Mineral Oil (Cat.# DY1151 or equivalent)
• 0.5 ml or 0.2 ml microcentrifuge tubes (compatible with thermal cycler)
• 1.5 ml microcentrifuge tubes
• BSA Fraction V (optional)
• aerosol-resistant pipette tips
• crushed ice
The CTT multiplex and GenePrint® Sex Identification System, Amelogenin are optimized for use with GeneAmp® reaction tubes and the Perkin-Elmer model 480 thermal
cycler. When using a thermal cycler on which a system was not optimized, there may
be a loss in product yield or sensitivity, and the balance between loci may change
slightly. Meticulous care must be taken to ensure successful amplification. See our
Web site or contact Technical Services for help optimizing amplification conditions.
Amplification Setup
We highly recommend that you wear gloves and use aerosol-resistant pipet tips to
prevent contamination.
1.
Thaw the STR 10X Buffer and 10X Primer Pairs, and place on ice.
Note: Mix reagents by vortexing for 15 seconds before each use.
2.
Place one clean, autoclaved 0.5 ml reaction tube for each reaction into a
rack, and label appropriately.
3.
Determine the number of reactions to be set up. This should include a positive and negative control reaction. Add 1 or 2 reactions to this number to
compensate for pipetting error. While this approach does consume a small
amount of each reagent, it ensures that you will have enough PCR maste
mix for all samples.
4.
Calculate the required amount of each component of the PCR master mix
(Table 2). Multiply the volume (µl) per sample by the total number of reactions (from Step 3) to obtain the final volume (µl).
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III.B. Amplification (continued)
5.
In the order listed in Table 2, add the final volume of each reagent to a
sterile tube. Mix gently (do not vortex), and place on ice.
Table 2. Combined CTTv Multiplex and Amelogenin Reactions
PCR Master Mix
Component
Volume Per Sample
(µl)
sterile water
14.85
STR 10X Buffer
CTT Multiplex 10X
Primer Pair
2.50
Amelogenin 10X
Primer Pair
Taq DNA Polymerase
(5u/µl)
Total Volume
Number of Reactions
Final Volume (µl)
2.50
2.50
0.15 (0.75u)
22.50
Note: The volume given assumes a Taq DNA polymerase concentration of
5 u/µl. For different enzyme concentrations, the volume of enzyme addedmust be adjusted accordingly. If the final volume of Taq DNA polymerase
added to the master mix is less than 0.5 µl, you may wish to dilute the
enzyme with STR 1X Buffer, and add a larger volume. The amount of sterile
water should be adjusted accordingly so that the final volume per reaction is
25 µl. Do not store diluted Taq DNA polymerase.
6.
Add 22.5 µl of PCR master mix to each tube, and place on ice. Failure to
keep the reagents and samples on ice can produce imbalanced amplification of multiplexed loci.
7.
Pipet 2.5 µl of each sample into the respective tube containing 22.5 µl of
PCR master mix.
8.
For the positive amplification control, pipet 2.5 µl (5ng) of K562 DNA (diluted
to 2 ng/µl) into a 0.5ml reaction tube containing 22.5 µl of PCR master mix.
9.
For a negative amplification control, pipet 2.5 µl of sterile water (instead of
template DNA) into a 0.5ml reaction tube containing 22.5 µl of PCR master
mix.
10. If you are using a thermal cycler with an unheated lid, add 1 drop of mineral
oil to each tube. Close the tubes.
Note: Allow the mineral oil to flow down the side of the tube and form an
overlay to limit sample loss or cross-contamination due to splattering.
11. Centrifuge the samples briefly to bring the contents to the bottom of the
tube.
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Thermal Cycling Protocol
1.
Place the tubes in a thermal cycler.
2.
Run the protocol below:
Initial Incubation:
96 °C for 2 minutes
Programmed Ramp Times: None
First 10 Cycles
94 °C for 1 minute
64 °C for 1 minute
70 °C for 1.5 minutes
Programed Ramp Times: None
Last 20 Cycles
90 °C for 1 minute
64 °C for 1 minute
70 °C for 1.5 minutes
Extension Step: None
Hold at 4 °C.
3.
After completing the thermal cycling protocol, store the samples at –20 °C.
Note: Storing the amplification products at our above 4 °C may result in
degradation products.
III.C. Polyacrylamide Gel Preparation
Materials to Be Supplied by the User
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
40% acrylamide:bis (19:1) and TEMED
TBE Buffer, 10X (Cat.# V4251)
Ammonium Persulfate, 10% (Cat.# V3131)
Urea (Cat.# V3171)
bind silane (methacryloxypropyltrimethoxysilane)
Gel Slick® solution (Cambrex Cat.# 50640)
0.5% acetic acid in 95% ethanol
Nalgene® tissue culture filter (0.2 micron)
polyacrylamide gel electrophoresis apparatus for gels ≥ 30cm (e.g.,
SA32 or S2)
glass plates and side spacers for polyacrylamide gel ≥ 30cm
14 cm vinyl doublefine sharkstooth comb(s), 49 point, 0.4mm thick; or
square-tooth comb, 35 cm, 60 wells (cut in half for 30 wells/gel), 0.4 mm
thick (Owl Scientific Cat.# S2S-60A)
power supply
Liqui-Nox® detergent (Use of Liqui-Nox® detergent is extremely
important, because other kinds of detergent can build up on the glass
plates.)
clamps (e.g., large office binder clips)
diamond pencil for marking glass plates
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Notes
1.
Use a 4% gel for separation of the CTTv and amelogenin loci.
2.
Unpolymerized acrylamide is a neurotoxin and suspected carcinogen; avoid
inhalation and contact with skin. Read the warning label, and take the necessary precautions when handling this substance. Always wear gloves and
safety glasses when working with acrylamide powder or solutions.
3.
Bind silane is toxic and should be used in a chemical fume hood.
4.
The longer glass plate will be treated with Gel Slick® solution to prevent the
gel from sticking, and the shorter glass plate will be treated with bind silane
to bind the gel. The two plates must be kept apart at all times to prevent
cross-contamination.
5.
All cleaning utensils (sponges) for the longer glass plates should be kept
separate from those for the shorter glass plates to prevent cross contamination of the binding solution.
6.
The shorter glass plate preparation must be repeated for each gel. The
longer glass plate preparation must be repeated after every four gels.
7.
To remove the glass plate treatments (Gel Slick® solution or bind silane)
immerse the plate(s) in 10% NaOH solution for 1 hour. Thoroughly rinse the
plate(s) with deionized water, and clean with a detergent. The same 10%
NaOH solution may be used for multiple gels.
8.
New glass plates should be soaked in 10% NaOH for 1 hour, then rinsed
thoroughly with deionized water before use. New plates also should be
etched with a diamond pencil in the corner of one side to distinguish the
sides of the plates in contact with the gel.
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Procedure
The following protocol is for the preparation of a denaturing polyacrylamide gel with
the dimensions of 31.0 cm wide × 38.5 cm high × 0.4 mm thick (e.g., S2 sequencing
gel electrophoresis apparatus, Whatman Cat.# 21105-010). Use one-half of the volumes described here for a gel with the dimensions of 17 cm wide × 32 cm high ×
0.4 mm thick (e.g., SA32 sequencing gel apparatus, Whatman Cat.# 31096-019).
1.
Thoroughly clean the shorter and longer glass plates twice with 95% ethanol
and Kimwipes® tissues.
Note: The gel side is the etched side of the glass plate.
2.
Using gloves, apply 3 ml of Gel Slick® solution onto the etched side of the
longer glass plate. With a dry paper towel, spread the Gel Slick® solution
using a circular motion over the entire surface.
3.
Wait 5 minutes for the Gel Slick® solution to dry. Remove the excess Gel
Slick® solution with a paper towel saturated with deionized water. Finally, dry
the glass plate with Kimwipes® tissue.
4.
In a chemical fume hood, prepare fresh binding solution by adding 3 µl of
bind silane to 1 ml of 0.5% acetic acid in 95% ethanol in a 1.5 ml tube. Wipe
the etched side of the shorter glass plate using a Kimwipes® tissue saturated with the freshly prepared binding solution. Be certain to wipe the entire
plate surface with the saturated tissue.
5.
Wait 5 minutes for the binding solution to dry. Wipe the shorter glass plate
3–4 times with 95% ethanol and Kimwipes® tissues to remove the excess
binding solution. Failure to wipe excess binding solution from the shorter
glass plate will cause the gel to stick to both plates, and the gel will be
destroyed upon separation of the glass plates after electrophoresis.
6.
Take special care not to allow the treated surfaces to touch each other.
Assemble the glass plates by placing 0.4 mm side spacers and a 0.4 mm
bottom spacer (optional) between the plates and using clamps to hold them
in place. Lean the assembled plates against a test tube rack or other similar
support.
7.
Prepare a 4% acrylamide solution (total of 75 ml) by combining the ingredients listed below:
Urea
31.50 g
deionized water
40.00 ml
TBE Buffer, 10X
3.75 ml
40% acrylamide:bis (19:1) 7.50 ml
total volume
75 ml
8.
Filter the acrylamide solution through a 0.2 micron filter (e.g., Nalgene®
tissue culture filter).
9.
Pour the filtered acrylamide solution into a squeeze bottle.
10. Add 50 µl of TEMED and 500 µl of 10% ammonium persulfate to the
acrylamide solution, and mix gently.
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11. Carefully pour the acrylamide solution between the glass plates. To prevent
bubble formation, start pouring at one side of the assembled plates and
maintain a constant flow of solution.
12. Position the gel horizontally, resting it on two test tube racks or other similar
supports. Remove any bubbles that may have formed.
13. Insert one or two 14 cm doublefine (49 point) sharkstooth combs, straight
side into the gel, between the glass plates (6 mm of the comb should be
between the two glass plates). If using a square-tooth comb, insert the
comb between the glass plates until the teeth are almost completely
inserted into the gel.
14. Secure the comb(s) with 2 to 3 clamps each.
15. Pour the remaining acrylamide solution into a disposable conical tube as a
polymerization control. Rinse the squeeze bottle, including the spout, with
water.
16. Allow polymerization to proceed for at least 1 hour. Check the polymerization control to be sure that polymerization has occurred.
Note: The gel may be stored overnight if a paper towel saturated with deionized water and plastic wrap are placed around the well end of the gel to prevent the gel from drying out. If no bottom spacer is used, the bottom of the
gel should be wrapped.
III.D.
Polyacrylamide Gel Electrophoresis
Gel Pre-Run
1.
Remove the clamps from the polymerized acrylamide gel, and clean the
glass plates with paper towels saturated with deionized water.
2.
Shave any excess polyacrylamide away from the comb. Remove the comb
and bottom spacer.
3.
Add 0.5X TBE to the bottom chamber of the electrophoresis apparatus.
4.
Gently lower the gel and glass plates into the buffer with the longer plate
facing out and the well side on top.
5.
Secure the glass plates to the sequencing gel apparatus.
6.
Add 0.5X TBE to the top buffer chamber of the electrophoresis apparatus.
7.
Using a 50–100 cc syringe filled with buffer, remove the air bubbles on the
top of the gel. Be certain the well area is devoid of air bubbles and small
pieces of polyacrylamide. Use a syringe with a bent 19-gauge needle to
remove the air bubbles between the glass plates on the bottom of the gel.
8.
Pre-run the gel to achieve a gel surface temperature of approximately 50 °C.
Consult the manufacturer’s instruction manual for the recommended electrophoresis conditions.
Note: As a reference, we generally use 60–65 watts for a 40 cm polyacrylamide gel 40–45 watts for a 32 cm gel. The gel running conditions may
have to be adjusted to reach a temperature of 50 °C.
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Sample Preparation
1.
Prepare the PCR samples by mixing 2.5 µl of each sample with 2.5 µl of
STR 2X Loading Solution.
Note: The sample alleles may appear more intense than ladder alleles on
the gel, but this should not interfere with allele determination. For more even
band intensities, mix 1 µl of each sample with 4 µl of a premix containing
2.5 µl of STR 2X Loading Solution and 1.5 µl of STR 1X Buffer.
2.
Add 2.5 µl (50 ng) of pGEM® DNA Markers to 2.5 µl of STR 2X Loading
Solution for each marker lane.
Note: We recommend loading pGEM® DNA Markers into the first and last
lanes of the gel.
3.
4.
Combine 2.0 µl of the CTT Allelic Ladder and 2.0 µl of Amelogenin ladder.
Mix well then combine 2.5 µl of this mixture with 2.5 µl of STR 2X Loading
Solution for each allelic ladder lane. The number of allelic ladder lanes used
depends on personal preference.
Briefly centrifuge the samples in a microcentrifuge to bring the contents to
the bottom of the tube.
Sample Loading
1.
Denature the samples by heating at 95 °C for 2 minutes, then immediately
chill on crushed ice or in an ice-water bath.
Note: Denature the samples just prior to loading the instrument.
2.
After the pre-run, use a 50–100 cc syringe filled with buffer to flush the urea
from the well area. If using a sharkstooth comb, carefully insert the comb
teeth into the gel approximately 1–2 mm. Leave the comb inserted in the gel
during both gel loading and electrophoresis.
3.
Load 3 µl of each sample into the respective wells. The loading process
should take no longer than 20 minutes to prevent the gel from cooling.
Gel Electrophoresis
1.
Once loading is complete, run the gel using the same conditions as for the
gel pre-run.
Note: In a 4% gel, bromophenol blue migrates at approximately 40 bases
and xylene cyanol migrates at approximately 170 bases.
2.
Knowing the size ranges for each locus (Table 4) and migration characteristics of the dyes (Step 1, above), stop electrophoresis any time after the
locus of interest has passed the midpoint of the gel. If running more than
one locus or a multiplex, be careful not to run the TH01 locus off the bottom
of the gel.
3.
Proceed to silver stain detection.
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Precast Polyacrylamide Gels
Because so many components of polyacrylamide gels are toxins, you may wish to use
precast gels. Most precast gels that are sold specifically for separation of DNA fragments and silver stain detection are designed to run on a specific apparatus. Therefore,
to use precast gels, you may need to also purchase an appropriate gel electrophoresis
apparatus. Several biotechnology supply companies sell such systems.
Invitrogen sells Novex® precast DNA retardation gels, and they specify the size
range that you can expect to separate when you use these gels. Their gels are
designed to run on the XCell SureLock™ MiniCell apparatus.
GE Life Sciences sells the GenePhor™ DNA Separation System and precast gels.
III.E.
Silver Staining
This protocol describes the use of the SILVER SEQUENCE™. Staining Reagents (Cat.#
Q4132). One system contains sufficient reagents to stain 10 sequencing size gels and
Materials to Be Supplied by the User
• SILVER SEQUENCE™ Staining Reagents (Cat.# Q4132)
• fix/stop solution
• staining solution
• developer solution (chilled to 4–10 °C)
• Nalgene® wash tubs (54.1 × 43.5 × 13 cm or appropriate size for your system)
• orbital shaker or rocker platform
fix/stop solution
10%
glacial acetic acid
staining solution
silver nitrate (AgNO3)
formaldehyde (HCOH)
(1.5 ml of 37% HCOH/liter)
1 g/L
0.056%
developing solution
30 g/L
0.056 M
2 mg/L
sodium carbonate (Na2CO3)
formaldehyde (HCOH)(1.5 ml of
37% HCOH/liter)
sodium thiosulfate(Na2S2O3 •
5H2O)
Use 2 liters of each solution per gel for each step (for a 54.1 × 43.5 × 13 cm tray).
Procedure
1.
After electrophoresis, empty the buffer chambers and carefully loosen the
gel clamps. Remove the glass plates from the apparatus.
2.
Place the gel and glass plates on a flat surface. Remove the comb and side
spacers. Use a plastic wedge to carefully separate the two glass plates. The
gel should be strongly affixed to the shorter glass plate.
3.
Place the gel (attached to the shorter plate) in a shallow plastic tray (e.g.,
Nalgene® wash tub).
4.
To silver stain, follow Steps a–h below. Gently agitate during each step.
Steps involving solutions containing formaldehyde should be performed in a
chemical hood.
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Step
Solution
Time
a.
fix/stop solution (See Note 1)
20 minutes
b.
deionized water
2 minutes
c.
repeat Step b, twice
2 × 2 minutes
d.
staining solution
30 minutes
e.
deionized water (See Note 2)
10 seconds
f.
developer solution
up to 5 minutes (untill alleles and
ladders are visible)
g.
fix/stop solution (See Note 3)
5 minutes
h.
deionized water
2 minutes
Notes:
1.
Save the fix/stop solution from Step 4a, to use in Step 4g.
2.
The duration of Step 4e is important. The total time from immersion in
deionized water to immersion in developer solution should be less than
20 seconds. If the deionized water rinse step does exceed 20 seconds,
repeat Step 4d.
3.
Add fix/stop solution directly to developer solution to stop developing reaction.
4.
Position the gel and shorter plate upright, and allow it to dry overnight. For
best results, the gel should be completely dried before APC Film development (Section III.F). Alternatively, to create film prints of the gel immediately,
cover the gel with plastic wrap, and expose your film.
Reusing Glass Plates
1.
Immerse the plate and affixed gel in a 10% NaOH solution for 1 hour to
overnight. Discard the gel, and clean the glass plate with deionized water
and a detergent such as Liqui-Nox® detergent. The 10% NaOH
solution may be reused for additional gels.
2.
All cleaning utensils and sponges for the longer glass plates should be kept
separate from those for the shorter glass plates to prevent cross-contamination of the binding solution.
III.F. Generating Film Images
A direct image may be produced using Automatic Processor Compatible (APC) Film. The
image produced on APC Film is the mirror image of the gel. Use of film allows the generation of multiple permanent images with more control over band and background intensity than does development of the gel alone. Handle all plates with gloved hands to avoid
fingerprints.
Materials to Be Supplied by the User
• white light box
• automatic film processor or film developing tanks
• Automatic Processor Compatible (APC) Film (Cat.# Q4411)
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1.
In the darkroom with a safelight on, place the dry, stained gel attached to
the shorter plate (gel side up) on a white fluorescent light box.
Note: For best results, the gel should be completely dry before the image is
captured with APC film. If capturing an image from a gel that has not been
dried, cover the gel with plastic wrap.
2.
Position the APC Film, emulsion side down, over the gel to be copied.
Note: The emulsion side of the film can be identified as the glossy white
surface; the nonemulsion side has a gray tint.
3.
Place a clean glass plate on top of the film to maintain contact between the
gel and film. Turn on the white light box, and expose the film for 1–2 minutes, depending on the gel background level and the intensity of the white
light. (This step must be optimized for individual light boxes.)
4.
Develop the film as recommended by the manufacturer. APC film may be
processed manually or with an automatic film processor. For automatic film
processors, follow the manufacturer’s instructions.
Note: The image produced on APC Film is the mirror image of the gel.
5.
If there is very little signal, decrease the exposure time used in Step 3. If the
film appears brown or black, increase the exposure time.
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