Complete Protocol

Complete Protocol
TECHNICAL MANUAL
PowerPlex® 16 BIO System
Instructions for Use of Product
DC6540
Revised 7/15
TMD016
PowerPlex® 16 BIO System
All technical literature is available at: www.promega.com/protocols/
Visit the web site to verify that you are using the most current version of this Technical Manual.
E-mail Promega Technical Services if you have questions on use of this system: [email protected]
1. Description.......................................................................................................................................... 2
2. Product Components and Storage Conditions......................................................................................... 3
3. Before You Begin.................................................................................................................................. 4
4. Protocols for DNA Amplification Using the PowerPlex® 16 BIO System.................................................... 5
4.A. Amplification Setup..................................................................................................................... 5
4.B. Amplification Thermal Cycling...................................................................................................... 7
5. Detection of Amplified Fragments Using the Hitachi FMBIO® II Fluorescence Imaging System.................. 9
5.A. Polyacrylamide Gel Preparation.................................................................................................... 9
5.B. Gel Pre-Run.............................................................................................................................. 11
5.C. Sample Preparation and Loading................................................................................................ 12
5.D. Gel Electrophoresis................................................................................................................... 13
5.E.Detection.................................................................................................................................. 13
6. Data Analysis..................................................................................................................................... 15
6.A. Background Adjustment............................................................................................................. 15
6.B. Color Separation Using Individual Bands.................................................................................... 16
6.C. Color Separation Using Bands In a Single Lane............................................................................ 16
6.D.Autobanding............................................................................................................................. 17
6.E.Controls.................................................................................................................................... 17
6.F. Allelic Ladders.......................................................................................................................... 18
6.G.Results..................................................................................................................................... 18
7. Troubleshooting................................................................................................................................ 21
8. References......................................................................................................................................... 25
9. Appendix........................................................................................................................................... 27
9.A. Advantages of Using the Loci in the PowerPlex® 16 BIO System.................................................... 27
9.B. Power of Discrimination............................................................................................................ 31
9.C. DNA Extraction and Quantitation Methods and Automation Support............................................. 32
9.D. The Internal Lane Standard 600 BIO........................................................................................... 32
9.E. Preparing the PowerPlex® 16 BIO System PCR Amplification Mix................................................. 33
9.F. Composition of Buffers and Solutions.......................................................................................... 34
9.G. Related Products....................................................................................................................... 35
9.H. Summary of Changes................................................................................................................. 36
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1
1.Description
STR (short tandem repeat) loci consist of short, repetitive sequence elements 3–7 base pairs in length (1–4). These
repeats are well distributed throughout the human genome and are a rich source of highly polymorphic markers, which
may be detected using the polymerase chain reaction (5–8). Alleles of STR loci are differentiated by the number of
copies of the repeat sequence contained within the amplified region and are distinguished from one another using
fluorescence detection following electrophoretic separation.
The PowerPlex® 16 BIO System(a–e) is used for human identification applications including forensic analysis, relationship testing and research use. The system allows coamplification and three-color detection of sixteen loci (fifteen STR
loci and Amelogenin). The PowerPlex® 16 BIO System contains the loci Penta E, D18S51, D21S11, TH01, D3S1358,
FGA, TPOX, D8S1179, vWA, Amelogenin, Penta D, CSF1PO, D16S539, D7S820, D13S317 and D5S818. In the
system, one primer specific for Penta E, D18S51, D21S11, TH01 and D3S1358 is labeled with fluorescein (FL); one
primer specific for FGA, TPOX, D8S1179, vWA and Amelogenin is labeled with Rhodamine Red™-X; and one primer
specific for Penta D, CSF1PO, D16S539, D7S820, D13S317 and D5S818 is labeled with 6-carboxy-4´,5´-dichloro2´,7´-dimethoxy-fluorescein (JOE). All sixteen loci are amplified simultaneously in a single tube and analyzed in a
single gel lane.
The PowerPlex® 16 Monoplex System, Penta E (Fluorescein) (Cat.# DC6591) and PowerPlex® 16 Monoplex System,
Penta D (JOE) (Cat.# DC6651) are available to amplify the Penta E and Penta D loci, respectively. Each monoplex
system allows amplification of a single locus to confirm results obtained with the PowerPlex® 16 System, PowerPlex®
16 BIO System or PowerPlex® 2.1 System. The monoplex systems also can be used to re-amplify DNA samples when
one or more of the loci do not amplify initially due to nonoptimal amplification conditions or poor DNA quality.
The PowerPlex® 16 BIO System is designed specifically for use with the Hitachi FMBIO® II Fluorescence Imaging
System. It provides all of the materials necessary to amplify STR regions of purified human genomic DNA except for
AmpliTaq Gold® DNA polymerase. This manual contains separate protocols for use of the PowerPlex® 16 BIO System
with the Perkin-Elmer model 480 and GeneAmp® PCR system 9600, 9700 and 2400 thermal cyclers in addition to
protocols for separation and detection of amplified materials.
Information about other Promega fluorescent STR systems is available upon request from Promega or online at:
www.promega.com/geneticidentity/
2
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2.
Product Components and Storage Conditions
PRODUCT
PowerPlex® 16 BIO System
S I Z E C A T. #
400 reactions
DC6540
Not For Medical Diagnostic Use. Cat.# DC6540 contains sufficient reagents for 400 reactions of 25µl each.
Includes:
Pre-amplification Components Box (Blue Label)
• 4 × 300µl Gold STHR 10X Buffer
• 4 × 250µl PowerPlex® 16 BIO 10X Primer Pair Mix
•
25µl 2800M Control DNA, 10ng/µl
Post-amplification Components Box (Beige Label)
• 4 × 125µl PowerPlex® 16 BIO Allelic Ladder Mix
•
100µl Matrix 16 BIO
• 2 × 300µl Internal Lane Standard (ILS) 600 BIO
• 2 × 1ml Bromophenol Blue Loading Solution
•
250µl Gel Tracking Dye
The PowerPlex® 16 BIO Allelic Ladder Mix is provided in a separate, sealed bag for shipping. This component should
be moved to the post-amplification box after opening.
Storage Conditions: Store all components except the 2800M Control DNA at –30°C to –10°C in a nonfrost-free
freezer. Store the 2800M Control DNA at 2 to 10°C. The PowerPlex® 16 BIO 10X Primer Pair Mix, PowerPlex® 16
BIO Allelic Ladder Mix, Matrix 16 BIO and Internal Lane Standard 600 BIO are light-sensitive and must be stored in
the dark. We strongly recommend that pre-amplification and post-amplification reagents be stored and used separately
with different pipettes, tube racks, etc.
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3
3.
Before You Begin
The application of PCR-based typing for forensic or paternity casework requires validation studies and quality-control
measures that are not contained in this manual (9,10). The quality of the purified DNA, as well as small changes in
buffers, ionic strength, primer concentrations, choice of thermal cycler and thermal cycling conditions, can affect
PCR success. We suggest strict adherence to recommended procedures for amplification, as well as for electrophoresis
and fluorescence detection. Additional research and validation are required if any modifications are made to the
recommended protocols.
PCR-based STR analysis is subject to contamination by minute amounts of human DNA. Extreme care should be taken
to avoid cross-contamination when preparing sample DNA, handling primer pairs, assembling amplification reactions
and analyzing amplification products. Reagents and materials used prior to amplification (Gold STHR 10X Buffer,
2800M Control DNA and PowerPlex® 16 BIO 10X Primer Pair Mix) are provided in a separate box and should be
stored separately from those used following amplification (PowerPlex® 16 BIO Allelic Ladder Mix, Internal Lane
Standard 600 BIO, Matrix 16 BIO, Bromophenol Blue Loading Solution and Gel Tracking Dye). 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 (e.g., ART® tips, Section 9.G).
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.
Reagents
Hazards
acrylamide
suspected carcinogen, toxic
ammonium persulfate
oxidizer, corrosive
formamide
(contained in the Bromophenol Blue Loading Solution)
irritant, teratogen
TEMED
corrosive, flammable
urea
irritant
4
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4.
Protocols for DNA Amplification Using the PowerPlex® 16 BIO System
Materials to Be Supplied by the User
•
model 480 or GeneAmp® system 9600, 9700 or 2400 thermal cycler (Applied Biosystems)
•microcentrifuge
•
0.5ml or 0.2ml (thin-walled) microcentrifuge tubes, MicroAmp® optical 96-well reaction plate or
MicroAmp® 8-strip reaction tubes (Applied Biosystems)
•
aerosol-resistant pipette tips (see Section 9.G)
•
AmpliTaq Gold® DNA polymerase (Applied Biosystems)
•
Nuclease-Free Water (Cat.# P1193)
•
Mineral Oil (Cat.# DY1151, for use with the thermal cycler model 480)
Note: If using the GeneAmp® PCR system 9600, 9700 or 2400 thermal cyclers, use 0.2ml MicroAmp® 8-strip reaction
tubes or MicroAmp® plate. For the Perkin-Elmer model 480, we recommend 0.5ml GeneAmp® thin-walled reaction
tubes.
We routinely amplify 0.5–1ng of template DNA in a 25µl reaction volume using the protocols detailed below. Expect to
see more intense bands for smaller loci and less intense bands for larger loci if more than the recommended amount of
template is used. Reduce the amount of template DNA or the number of cycles to correct this.
The PowerPlex® 16 BIO System is optimized for the GeneAmp® PCR system 9600 thermal cycler. Amplification
protocols for the GeneAmp® PCR systems 9700 and 2400 thermal cyclers and Perkin-Elmer model 480 thermal cycler
are provided.
4.A. Amplification Setup
The use of gloves and aerosol-resistant pipette tips is highly recommended to prevent cross-contamination. Keep all
pre-amplification and post-amplification reagents in separate rooms. Prepare amplification reactions in a room
dedicated for reaction setup. Use equipment and supplies dedicated for amplification setup.
Meticulous care must be taken to ensure successful amplification. A guide to amplification troubleshooting is
provided in Section 7.
!
The concentration of 2800M Control DNA was determined by measuring absorbance at 260nm. Quantification of this
control DNA by other methods, such as qPCR, may result in a different value. Prepare a fresh DNA dilution for each set
of amplifications. Do not store diluted DNA (e.g., 0.25ng/μl or less).
1.
Thaw Gold STHR 10X Buffer and PowerPlex® 16 BIO 10X Primer Pair Mix.
Notes:
1.
Mix reagents by vortexing for 15 seconds before each use. Do not centrifuge the 10X Primer Pair Mix after
vortexing, as this may cause the primers to be concentrated at the bottom of the tube.
2.
A precipitate may form in Gold STHR 10X Buffer. If this occurs, warm the buffer briefly at 37°C, then
vortex until the precipitate is in solution.
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4.A. Amplification Setup (continued)
2.
Determine the number of reactions to be set up. This should include positive and negative control reactions.
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 amplification mix for all samples. It also
ensures that each reaction contains the same PCR amplification mix.
3.
Place one clean, 0.2ml or 0.5ml reaction tube for each reaction into a rack, and label appropriately. Alternatively,
use a MicroAmp® plate, and label appropriately.
4.
Add the final volume of each reagent listed in Table 2 into a sterile tube. Mix gently.
Table 2 shows the component volumes per reaction. A worksheet to calculate the required amount of each
PCR amplification mix component is provided in Section 9.E (Table 10).
Table 2. PCR Amplification Mix for the PowerPlex® 16 BIO System.
PCR Amplification Mix Component1
nuclease-free water
to a final volume of 25.0µl
2.5µl
Gold STHR 10X Buffer
PowerPlex® 16 BIO 10X Primer Pair Mix
AmpliTaq Gold DNA polymerase
®
2
template DNA (0.5–1ng)
3
total reaction volume
Volume Per Sample
2.5µl
0.8µl (4u)
up to 19.2µl
25µl
Add nuclease-free water to the PCR amplification mix first; then add Gold STHR 10X Buffer,
PowerPlex® 16 BIO 10X Primer Pair Mix and AmpliTaq Gold® DNA polymerase. The
template DNA will be added at Step 6.
1
2
Assumes AmpliTaq Gold® DNA polymerase is at 5u/µl. If the concentration is different, the
volume of enzyme used must be adjusted accordingly.
Store DNA templates in nuclease-free water or TE–4 buffer (10mM Tris-HCl [pH 8.0],
0.1mM EDTA). If the DNA template is stored in TE buffer that is not pH 8.0 or contains a
higher EDTA concentration, the volume of DNA added should not exceed 20% of the final
reaction volume. Amplification efficiency and quality can be greatly altered by changes in pH
(due to added Tris-HCl), available magnesium concentration (due to chelation by EDTA), or
other PCR inhibitors, which may be present at low concentrations depending on the source of
the template DNA and the extraction procedure used.
3
!
6
Amplification of >2ng of DNA template results in an imbalance in band intensities from locus to locus.
The smaller loci show greater amplification yield than larger loci. Reducing the number of cycles in the
amplification program by 2 to 4 cycles (i.e., 10/20 or 10/18 cycling) can improve locus-to-locus balance.
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5.
Pipet PCR amplification mix into each reaction tube.
6.
Pipet each template DNA (0.5–1ng) into the respective tube containing PCR amplification mix.
7.
For the positive amplification control, vortex the tube of 2800M Control DNA; then dilute an aliquot to
0.5ng or 1ng in the desired template DNA volume. Pipet 0.5–1ng of the diluted DNA into a microcentrifuge
tube containing PCR amplification mix.
Note: To store diluted 2800M Control DNA, dilute the DNA to 0.5ng/μl in TE–4 buffer with 20μg/ml glycogen
and store at 4°C. Do not store dilutions performed in water.
8.
For the negative amplification control, pipet nuclease-free water or TE–4 buffer instead of template DNA into a
reaction tube containing PCR amplification mix.
9.
If using the GeneAmp® PCR System 9600, 9700 or 2400 thermal cycler and MicroAmp® reaction tubes or
plates, no addition of mineral oil to the reaction tubes is required. However, if using the model 480 thermal
cycler and GeneAmp® reaction tubes, add one drop of mineral oil to each tube before closing.
Note: Allow the mineral oil to flow down the side of the tube and form an overlay to limit sample loss or crosscontamination due to splattering.
10. Optional: Briefly centrifuge the tubes to bring contents to the bottom and remove any air bubbles.
4.B. Amplification Thermal Cycling
This manual contains protocols for use of the PowerPlex® 16 BIO System with the Perkin-Elmer model 480 and
GeneAmp® PCR system 9600, 9700 and 2400 thermal cyclers. For information about other thermal cyclers, please
contact Promega Technical Services by e-mail: [email protected]
Amplification and detection instrumentation may vary. Testing at Promega shows that 10/22 cycles work well for
0.5–1ng of purified DNA templates. For higher amounts of input DNA or to decrease sensitivity, fewer cycles, such as
10/16, 10/18 or 10/20, should be evaluated. In-house validation should be performed.
1.
Place the tubes or MicroAmp® plate in the thermal cycler.
2.
Select and run a recommended protocol. The preferred protocols for use with the GeneAmp® PCR system 9600,
9700 and 2400 and Perkin-Elmer model 480 thermal cyclers are provided below.
3.
After completion of the thermal cycling protocol, store amplified samples at –20°C in a light-protected box.
Note: Long-term storage of amplified samples at 4°C or higher may produce degradation products.
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7
Protocol for the GeneAmp® PCR
System 9700 Thermal Cycler1
Protocol for the GeneAmp® PCR
System 2400 Thermal Cycler
95°C for 11 minutes, then:
95°C for 11 minutes, then:
96°C for 1 minute, then:
96°C for 1 minute, then:
ramp 100% to 94°C for 30 seconds
ramp 29% to 60°C for 30 seconds
ramp 23% to 70°C for 45 seconds
for 10 cycles, then:
ramp 100% to 94°C for 30 seconds
ramp 100% to 60°C for 30 seconds
ramp 23% to 70°C for 45 seconds
for 10 cycles, then:
ramp 100% to 90°C for 30 seconds
ramp 29% to 60°C for 30 seconds
ramp 23% to 70°C for 45 seconds
for 22 cycles, then:
ramp 100% to 90°C for 30 seconds
ramp 100% to 60°C for 30 seconds
ramp 23% to 70°C for 45 seconds
for 22 cycles, then:
60°C for 30 minutes
60°C for 30 minutes
4°C soak
4°C soak
Protocol for the GeneAmp® PCR
System 9600 Thermal Cycler
Protocol for the Perkin-Elmer
Model 480 Thermal Cycler
95°C for 11 minutes, then:
95°C for 11 minutes, then:
96°C for 1 minute, then:
96°C for 2 minutes, then:
94°C for 30 seconds
ramp 68 seconds to 60°C (hold for 30 seconds)
ramp 50 seconds to 70°C (hold for 45 seconds)
for 10 cycles, then:
94°C for 1 minute
60°C for 1 minute
70°C for 1.5 minutes
for 10 cycles, then:
90°C for 30 seconds
ramp 60 seconds to 60°C (hold for 30 seconds)
ramp 50 seconds to 70°C (hold for 45 seconds)
for 22 cycles, then:
90°C for 1 minute
60°C for 1 minute
70°C for 1.5 minutes
for 22 cycles, then:
60°C for 30 minutes
60°C for 30 minutes
4°C soak
4°C soak
When using the GeneAmp® PCR System 9700 thermal cycler, the ramp rates indicated in the cycling program must be
set, and the program must be run in 9600 ramp mode.
1
The ramp rates are set in the ramp rate modification screen. While viewing the cycling program, navigate to the ramp
rate modification screen by selecting “More”, then “Modify”. On the ramp rate modification screen the default rates for
each step are 100%. The rate under each hold step is the rate at which the temperature will change to that hold
temperature. Figure 1 shows the ramp rates for the GeneAmp® PCR System 9700 thermal cycler.
The ramp mode is set after “start” has been selected for the thermal cycling run. A Select Method Options screen
appears. Select 9600 ramp mode, and enter the reaction volume.
8
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94.0°C
100%
60.0°C
29%
70.0°C
23%
3 tmp 22 cycles
90.0°C
100%
60.0°C
29%
70.0°C
23%
7486MA
3 tmp 10 cycles
Figure 1. The ramp rates for the GeneAmp® PCR System 9700 thermal cycler.
5.
Detection of Amplified Fragments Using the Hitachi FMBIO® II Fluorescence Imaging System
Materials to Be Supplied by the User
(Solution compositions are provided in Section 9.F.)
•
polyacrylamide gel electrophoresis apparatus
•
dry heating block, water bath or thermal cycler
•
power supply (4,000 volt)
•
squaretooth comb, 35cm, 60 wells (cut in half for 30 wells/gel), 0.4mm thick (Owl Scientific Cat.# S2S-60A)
•Nalgene® tissue culture filter (0.2 micron)
•
•
•
•
•
•
aerosol-resistant pipette tips (Section 9.G)
low-fluorescence glass plates: 43cm x 19cm x 0.4mm (The Gel Company, Cat.# GG047-B0505S)
spacers, 0.4mm clear spacers (The Gel Company, Cat.# SGR47-036)
SA-43 Extension (Lab Repco, Cat.# 31096423) for use with 43cm glass plates
clamps (e.g., large office binder clamps)
50% Long Ranger® gel solution (Cambrex Cat.# 50611), Long Ranger Singel® pack for ABI sequencers 377–36cm
(Cambrex Cat.# 50691) or PAGE-PLUS™ concentrate, 40% solution (Amresco, Inc., Cat.# E562)
•
TBE 10X buffer
•
10% Ammonium Persulfate (Cat.# V3131)
•
Urea (Cat.# V3171)
•TEMED
•
bind silane (methacryloxypropyltrimethoxysilane), for use with squaretooth combs
•Liqui-Nox® detergent
•
filter set for the PowerPlex® 16 BIO System (MiraiBio Cat.# 11999-246-00)
5.A. Polyacrylamide Gel Preparation
Acrylamide (Long Ranger® gel solution) 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 solutions.
!
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5.A. Polyacrylamide Gel Preparation (continued)
1.
Thoroughly clean glass plates twice with hot water and a 1% Liqui-Nox® solution. Rinse extremely well with
deionized water. Allow the glass plates to air-dry in a dust-free environment.
Dust particles will be detected in the scan using the 577 filter. Use lint-free paper (e.g., Kimwipes® tissues) to
clean the glass plates both before pouring the gel and prior to scanning. Air from a bulb may also be used to
remove dust.
!
If using a squaretooth comb, one of the glass plates requires bind silane treatment. The plates do not require
special silane treatment when using a sharkstooth comb.
Bind Silane Treatment of Glass Plate
Prepare fresh binding solution in a chemical fume hood by adding 3µl of bind silane to a 1.5ml microcentrifuge
tube containing 1.0ml of 0.5% acetic acid in 95% ethanol. Wipe the short plate with a Kimwipes® tissue saturated
with freshly prepared binding solution. Wait 5 minutes for the binding solution to dry. Wipe the comb area of the
glass plate 5–6 times with 95% ethanol and Kimwipes® tissues to remove excess binding solution.
2.
Assemble the glass plates by placing 0.4mm side spacers between the front and rear glass plates, using binder
clamps to hold them in place (3–4 clamps on each side). A bottom spacer is neither required nor recommended.
Place the assembly horizontally on a test tube rack or similar support.
Note: We recommend clear spacers. If white or opaque spacers are used, place black electrical tape on the longer
plate over the spacer area to prevent the spacers from being included in the scan, which can affect the signal and
background.
3.
Prepare a 5% Long Ranger® or 6% PAGE-PLUS™ acrylamide solution by combining the ingredients listed in
Table 3.
Table 3. Preparation of 5% Long Ranger® Polyacrylamide Gels.
Component
5% Gel
Final Concentration
urea
18.0g
6M
deionized water
26.0ml
—
5.0ml
1X
5%
10X TBE Buffer
50% Long Ranger gel solution
5.0ml
total volume
50ml
Component
®
1
6% PAGE-PLUS™ Gel
Final Concentration
urea
18g
6M
deionized water
24ml
—
10X TBE buffer
5ml
1X
40% PAGE-PLUS™ gel solution
7.5ml
6%
total volume
50ml
Long Ranger Singel Packs may also be used.
1
10
®
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4.
Filter the acrylamide solution through a 0.2 micron filter, and pour into a squeeze bottle.
5.
Add the appropriate amounts of TEMED and 10% ammonium persulfate to the acrylamide solution, and mix gently.
Component
5% Long Ranger® Gel (50ml)
TEMED
18.0g
10% ammonium persulfate
26.0ml
6.
Pour the gel by starting at the well end of the plates and carefully pouring the acrylamide between the horizontal
glass plates. Allow the solution to fill the top width of the plates. Slightly tilt the plates to assist movement of the
solution to the bottom of the plates while maintaining a constant flow of solution. When the solution begins to
flow out from the bottom, position the plates horizontally.
7.
Insert the squaretooth comb between the glass plates until the teeth are almost completely inserted into the gel,
or insert one 14cm doublefine (49 point) sharkstooth comb, straight side into the gel, between the glass plates
(6mm of the comb should be between the two glass plates).
8.
Secure the comb with three evenly spaced clamps.
9.
Pour the remaining acrylamide solution into a disposable conical tube as a polymerization control. Rinse the
squeeze bottle, including the spout, with water.
10. 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 top and bottom to prevent the gel from drying out (crystallization of the urea will destroy the gel).
5.B. Gel Pre-Run
1.
Remove the clamps from the polymerized acrylamide gel. If necessary, clean any excess acrylamide from the glass
plates with Kimwipes® tissues saturated with deionized water.
2.
Shave any excess polyacrylamide away from the comb; remove the comb.
3.
Add 1X TBE buffer to the bottom chamber of the electrophoresis apparatus.
4.
Gently lower the gel/glass plates unit into the buffer with the longer plate facing out and the well side on top.
5.
Secure the glass plates to the gel electrophoresis apparatus.
6.
Add 1X TBE buffer to the top chamber of the electrophoresis apparatus.
7.
Using a 50–100cc syringe filled with buffer, remove any air bubbles on the top of the gel. Be certain the well area
is devoid of air bubbles and small pieces of polyacrylamide. Using a syringe with a bent 18-gauge needle, remove
any air bubbles from 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 watts for 45–60 minutes for a 43cm gel. The gel running conditions
may need to be adjusted to reach a temperature of 50°C.
9.
Prepare samples and allelic ladder samples during the gel pre-run.
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5.C. Sample Preparation and Loading
The Internal Lane Standard 600 (ILS) BIO is included in the PowerPlex® 16 BIO System for use as an internal size
marker. With this approach, only 2–3 lanes of the PowerPlex® 16 BIO Allelic Ladder Mix are required per gel.
The Matrix 16 BIO is included with the system to aid in color separation. The Matrix 16 BIO should be run in one or
two lanes per gel.
1.
Prepare a loading cocktail by combining and mixing the ILS 600 BIO and Bromophenol Blue Loading Solution as
follows:
Volume Per
Sample
×
Internal Lane Standard BIO
1µl
×
Bromophenol Blue Loading Solution
3µl
×
Number of
Lanes
=
Total
Volume
2.
Vortex for 10–15 seconds.
3.
Combine 4µl of prepared loading cocktail and 2µl of amplified sample or PowerPlex® 16 BIO Allelic Ladder Mix.
Vortex the allelic ladder prior to pipetting.
4.
For matrix lanes, combine 2.5µl of Matrix 16 BIO and 2.5µl of Bromophenol Blue Loading Solution.
Note: If the fluorescent signal is too intense, dilute samples in Gold STHR 1X Buffer before mixing with loading
cocktail or use less DNA template in the amplification reactions.
5.
Optional: Place 2µl of Gel Tracking Dye and 3µl of Gold STHR 1X Buffer in one tube. The Gel Tracking Dye
contains both bromophenol blue and xylene cyanol. This dye is loaded in the outermost lane of the gel at least
2 lanes from the nearest sample and is used as a visual indicator of migration.
Note: The xylene cyanol dye in the Gel Tracking Dye will fluoresce when scanned with the Hitachi FMBIO® II
Fluorescence Imaging System. Leave at least two empty lanes between the Gel Tracking Dye and sample lanes.
6.
Briefly centrifuge samples to bring contents to the bottom of the tubes.
7.
Just prior to loading the gel, denature samples by heating at 95°C for 2 minutes, and immediately chill on
crushed ice or in an ice water bath.
8.
After the pre-run, use a 50–100cc syringe filled with buffer and fitted with a bent 18-gauge needle to flush urea
from the well area. If using a squaretooth comb, do not reinsert the comb, as the samples will be loaded directly
into the wells. If using a sharkstooth comb, insert the teeth into the gel approximately 1–2mm, and leave the
comb inserted in the gel during both gel loading and electrophoresis.
9.
Load 3µl of each denatured sample into the respective wells. The loading process should take no longer than
20 minutes to prevent the gel from cooling.
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5.D. Gel Electrophoresis
1.
After loading, run the gel using the same conditions as in Section 5.B. Observe the lane containing Gel Tracking
Dye to monitor sample migration. In a 5% Long Ranger® acrylamide gel, xylene cyanol dye migrates at approximately 190 bases, and in a 6% PAGE-PLUS™ acrylamide gel, xylene cyanol migrates at approximately 130 bases.
In both gels, bromophenol blue migrates at less than 80 bases.
2.
Based on size ranges for each locus (Table 7) and migration characteristics of the dyes contained in the Gel
Tracking Dye, stop electrophoresis before the smallest locus (i.e., Amelogenin) has reached the bottom of the gel.
Note: Some rare alleles (i.e., 9 repeats) for the D3S1358 locus are close to 100 bases in size. The gel should be
stopped before the 100-base fragment of the ILS 600 BIO has reached the bottom.
Table 4. Recommended Run Times for SA-43 Gels.
Leading Edge of Xylene Cyanol
(distance from bottom of gel)
Approximate Run Time
(60 Watts)
5% Long Ranger®
17.5cm
1 hour, 30 minutes
6% PAGE-PLUS™
9.5cm
2 hours
Gel Type
These recommendations are based on a one-hour pre-run. Run time and xylene cyanol
migration will vary with length of pre-run.
5.E.Detection
1.
After electrophoresis, remove the gel/glass plate unit from the apparatus. Do not separate the glass plates.
2.
The plates must be thoroughly cleaned before scanning. Thoroughly clean both sides of the gel/glass plate unit
with deionized water and lint-free paper before scanning. Ethanol should not be used to clean the plate unit;
ethanol fluoresces and may be detected as background by the FMBIO® instrument.
3.
The protocols in the data analysis section use the following filter setup:
Filter Holder 1 (Upper Filter)
1CH
598nm
3CH
505nm
Filter Holder 2 (Lower Filter)
4CH
577nm
13137MA
2CH
665nm
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5.E. Detection (continued)
4.
Scan the gel using the parameters listed in Table 5. Use the 598nm filter (Channel 1) to detect Rhodamine Red™-Xlabeled loci, the 665nm filter (Channel 2) to detect the Texas Red®-X-labeled ILS 600 BIO, the 505nm filter
(Channel 3) to detect the fluorescein-labeled fragments and the 577nm filter (Channel 4) to detect JOE-labeled
fragments.
Notes:
1.
Dust particles will be detected in the scan using the 577nm filter.
2.
The 598nm filter in Channel 1 is acceptable for autofocusing.
Table 5. Instrument Parameters for the Hitachi FMBIO® II Fluorescence Imaging System and
PowerPlex® 16 BIO System.
Parameter
Specification
Material Type
acrylamide gel
Resolution:Horizontal
Vertical
Rate
Repeat
150dpi
150dpi
NA
256 times
Gray Level Correction Type
range
Cutoff Threshold: Low (background)
High (signal)
50%
1%
Reading Sensitivity
Focusing Point1
100% (598nm channel)
100% (665nm channel)
100% (505nm channel)
100% (577nm channel)
0.0mm
NA = not applicable.
Focusing point of 0.0mm is based on use of 5mm glass plates. If using precast
gels or thinner glass plates, the focusing point may need to be adjusted. Optimal
focusing point may vary from instrument to instrument.
1
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6.
Data Analysis
Four-color separation requires FMBIO® Analysis Software Macintosh® Version 8.0 or higher. The procedures included
in this manual are suggestions for color separation. Further optimization may be needed for individual laboratories.
6.A. Background Adjustment
1.
Create a new project using the raw data files.
2.
Go to “Multi”, and switch the display mode to “over”.
3.
Enlarge the project window to display four channels.
Note: The initial multicolor image may not be displayed as four colors.
4.
Highlight and change the colors in the boxes so that Channel 1 is red, Channel 2 is blue, Channel 3 is green and
Channel 4 is yellow.
5.
Perform background adjustment prior to multicolor separation. Adjust the background for each channel (Figure 2).
a.
From the multicolor image, zoom in on an area of medium-intensity band to 200X or 300X.
b.
From the multicolor image, find a medium-intensity band in the channel (color) of interest.
c.
Switch to the black and white image for that channel.
d.
From the Gray Level Adjustment window, highlight the Low (Background) percent box.
e.
Draw a box in the area directly above and almost on top of the medium-intensity band (Figure 2). Select
“Set”. This often results in a background above 90%.
Note: Choose background while at 200–300X for each color.
f.
Do not adjust the signal. Signal adjustment should be performed only if the signal is unusually high. If
signal needs adjustment, highlight the High (Signal) percent box, then select a band of medium intensity
for signal adjustment. Select “Set”.
6.
Repeat Step 5 for each of the other three colors by starting at the colored image and choosing the appropriate
color band.
7.
Review the gel image in the Over Display Mode under “Multi”. Lanes containing the Matrix 16 BIO sample
should have this pattern repeated three times from the largest to smallest band: red, green, blue and yellow.
Note: Make sure that the channels are set as shown in Step 4.
8.
Perform color separation using the method described in Section 6.B or 6.C.
3484TA07_1A
Enlarged
view
Figure 2. Area of scan to choose for background adjustment.
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6.B. Color Separation Using Individual Bands
Perform the multicolor separation using individual bands from the Matrix 16 BIO lane(s). The Matrix 16 BIO sample
should have this pattern repeated three times from the largest to smallest band: red, green, blue and yellow.
1.
From the multicolor image, zoom in on matrix lane to 200X or 300X.
2.
Highlight “Color Separation” under “Multi”. A color separation window will be displayed, and the “Basis Image”
shown will be red.
3.
Draw a box around a red band (top band of the matrix), avoiding as much background as possible, and select
“Set”.
4.
Repeat Steps 2 and 3 for each of the other colors by changing the “Basis Image” to the color of the band that is
highlighted.
5.
Select “OK” to generate the separated images.
6.
Select “OK” when asked if you want to execute color separation.
6.C. Color Separation Using Bands In a Single Lane
The multicolor separation can be done using a single lane. Use the Matrix 16 BIO lane.
Read the section on image analysis tools in the FMBIO® user’s manual before creating a color separation matrix with
the Multi-Band Color Separator.
1.
Select “1 D-Gel” under Tools.
2.
Select “Layer”, and highlight “filename.2CH”. Select “OK”.
3.
Move the range to just above the top and just below the bottom of the matrix bands.
4.
Select the single lane tool
5.
Select “Autoband” on the side menu. Make sure that 12 bands are autobanded.
6.
Select “Color Separation” from the Multi menu, then select “Multi”. A Multi-Band Color Separator window will
be displayed.
7.
Select “Copy”, or highlight a stored pattern. The pattern should have three sets of four colors with the pattern:
red, green, blue and yellow.
8.
If the pattern is not displayed correctly, highlight the colored box under “Use”, and select the correct color.
Note: The Matrix 16 BIO pattern can be stored by highlighting “Save as” and typing in “Matrix 16 BIO”.
9.
Select “OK”.
from the side menu, and drag it down the matrix sample lane.
10. Select “OK” from the Multi-Band Color Separator window.
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6.D.Autobanding
1.
View and analyze the gel image to determine allele designations as recommended in the FMBIO® user’s manual.
For information regarding the use of FMBIO® Analysis Software, contact the Hitachi Technical Support Group
(510-337-2000 or [email protected]).
2.
We recommend the following autobanding parameters:
Parameter
3.
Default Setting
Recommended Settings
Starting Slope
2.5
1.5–2.0
Ending Slope
2.5
1.5–2.0
Duration
0.2
0.1
Noise Level
20
15–30
The data from the four-color analysis should contain the following:
Channel/Filter Display as (Color)
Dye Label
Loci
1/598nm
Red
Rhodamine Red™-X
FGA, TPOX, D8S1179, vWA, Amelogenin
2/665nm
Blue
Texas Red -X
Internal Lane Standard 600 BIO
3/505nm
Green
Fluorescein
Penta E, D18S51, D21S11, TH01, D3S1358
4/577nm
Yellow
JOE
Penta D, CSF1PO, D16S539, D7S820,
D13S317, D5S818
®
6.E.Controls
Observe the lanes containing the negative controls. Using the protocols defined in this manual, the negative controls
should be devoid of amplification products.
Observe the lanes containing the 2800M Control DNA. Compare the 2800M Control DNA allelic repeat sizes with the
locus-specific allelic ladder. The expected 2800M Control DNA allele designations for each locus are listed in Table 8
(Section 9.A).
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6.F. Allelic Ladders
In general, allelic ladders contain fragments of the same lengths as either most or all known alleles for the locus. Allelic
ladder sizes and repeat units are listed in Table 7 (Section 9.A). Visual comparison between allelic ladder and amplified
samples of the same locus allows precise assignment of alleles. Analysis using specific instrumentation also allows allele
determination by comparing amplified sample fragments with either allelic ladders, internal size standards or both (see
software documentation from instrument manufacturer). When using an internal lane standard, the calculated lengths
of allelic ladder components will differ from those listed in Table 7. This is due to differences in migration resulting
from sequence differences between allelic ladder fragments and internal lane standard fragments. Dye labels also affect
fragment migration.
Note: It may prove helpful to confirm that your gel analysis software identifies the correct number of alleles present in
the allelic ladder lanes prior to analysis of sample lanes. The PowerPlex® 16 BIO Allelic Ladder has 86 alleles in the
fluorescein channel (20 Penta E alleles, 22 D18S51 alleles, 25 D21S11 alleles, 10 TH01 alleles and 9 D3S1358 alleles),
61 alleles in the JOE channel (14 Penta D alleles, 10 CSF1PO alleles, 9 D16S539 alleles, 9 D7S820 alleles, 9 D13S317
alleles and 10 D5S818 alleles), and 55 alleles in the Rhodamine Red™-X channel (20 FGA alleles, 8 TPOX alleles, 12
D8S1179 alleles, 13 vWA alleles and 2 Amelogenin alleles).
6.G.Results
Representative results using the PowerPlex® 16 BIO System are shown in Figures 3 and 4.
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A. 505nm Scan:
Fluorescein
B. 577nm Scan:
JOE
L 1 2 3 4 5 L
Penta E
C.
598nm Scan:
Rhodamine Red™-X
L 1 2 3 4 5 L
L 1 2 3 4 5 L
Penta D
CSF1PO
FGA
D18S51
D16S539
TPOX
D7S820
D8S1179
D21S11
D13S317
TH01
vWA
D5S818
Amel.
6330TA
D3S1358
Figure 3. The PowerPlex® 16 BIO System. Five single-source DNA samples (lanes 1–5) were amplified with the
PowerPlex® 16 BIO Primer Pair Mix using 1ng of DNA template. Lane 1 shows the results using 1ng of 9947A DNA.
Lanes labeled L contain allelic ladders for each of the sixteen loci contained in the system. All materials were separated
using a 6% PAGE-PLUS™ acrylamide gel and detected using the Hitachi FMBIO® II Fluorescence Imaging System.
Panel A. A scan using a 505nm filter showing the fluorescein-labeled loci Penta E, D18S51, D21S11, TH01 and
D3S1358. Panel B. A scan using a 577nm filter showing the JOE-labeled loci Penta D, CSF1PO, D16S539, D7S820,
D13S317 and D5S818. Panel C. A scan using a 598nm filter showing the Rhodamine Red™-X-labeled loci FGA,
TPOX, D8S1179, vWA and Amelogenin.
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A. 505nm Scan:
Fluorescein
B. 577nm Scan:
JOE
L 1 2 3 4 5
C.
598nm Scan:
Rhodamine Red™-X
L12345
D. 665nm Scan:
Texas Red®-X
L12345
600
550
Penta E
Penta D
FGA
CSF1PO
D18S51
D16S539
500
475
450
425
400
375
350
TPOX
325
300
D7S820
D8S1179
275
250
D21S11
D13S317
225
200
TH01
180
vWA
160
D5S818
D3S1358
140
Amel.
100
6331TA
120
Figure 4. Amplification of various template amounts with the PowerPlex® 16 BIO System. A single-source
DNA template (2, 1, 0.5 or 0.2ng) was amplified. The results are shown in lanes 1–4, respectively. Lane 5 shows the
amplification results with no DNA template. Lanes labeled L contain the PowerPlex® 16 BIO Allelic Ladder. All
materials were separated using a 6% PAGE-PLUS™ acrylamide gel and detected using the Hitachi FMBIO® II
Fluorescence Imaging System. Panel A. A scan using a 505nm filter showing the fluorescein-labeled loci Penta E,
D18S51, D21S11, TH01 and D3S1358. Panel B. A scan using a 577nm filter showing the JOE-labeled loci Penta D,
CSF1PO, D16S539, D7S820, D13S317 and D5S818. Panel C. A scan using a 598nm filter,showing the Rhodamine
Red™-X-labeled loci FGA, TPOX, D8S1179, vWA and Amelogenin. Panel D. A scan using a 665nm filter, which
results in an image of the Texas Red®-X-labeled Internal Lane Standard 600 BIO.
20
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7.Troubleshooting
For questions not addressed here, please contact your local Promega Branch Office or Distributor. Contact information
available at: www.promega.com. E-mail: [email protected]
Symptoms
Causes and Comments
Faint or absent allele bandsImpure template DNA. Because of the small amount of template
used, this is rarely a problem. Depending on the DNA extraction
procedure used and sample source, inhibitors may be present in
the DNA sample. Diluting the template in TE–4 buffer or water
prior to amplification may improve results.
Insufficient template DNA. Use the recommended amount of
template DNA.
Insufficient enzyme activity. Use the recommended amount of
AmpliTaq Gold® DNA polymerase. Check the expiration date on
the tube label.
Incorrect amplification program. Confirm the amplification
program.
High salt concentration or altered pH. If the DNA template is
stored in TE buffer that is not pH 8.0 or contains a higher EDTA
concentration, the DNA volume should not exceed 20% of the
total reaction volume. Carryover of K+, Na+, Mg2+ or EDTA from
the DNA sample can negatively affect PCR. A change in pH also
may affect PCR. Store DNA in TE–4 buffer (10mM Tris-HCl [pH
8.0], 0.1mM EDTA) or nuclease-free water.
Primer concentration was too low. Use the recommended primer
concentration. Vortex the PowerPlex® 16 BIO 10X Primer Pair
Mix for 15 seconds before use.
Thermal cycler, plate or tube problems. Review the thermal
cycling protocols in Section 4.B. We have not tested other
reaction tubes, plates or thermal cyclers. Calibrate the thermal
cycler heating block if necessary.
Samples were not denatured completely. Heat-denature samples
at 95°C for 2 minutes, and immediately chill on crushed ice or in
an ice water bath prior to loading the gel. Do not cool the
samples in a thermal cycler set at 4°C, as this may lead to
artifacts due to DNA re-annealing.
Background level was too high (≥99%) in the black and white
image in question. Repeat the color separation process starting
with the gray level adjustment and using raw data, OR lower the
background to a percentage equivalent to the percentage that
was set in the other three images.
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7.
Troubleshooting (continued)
Symptoms
Causes and Comments
Bands are fuzzy throughout the lanesPoor-quality polyacrylamide gel. Prepare acrylamide and buffer
solutions using high-quality reagents. We recommend 5% Long
Ranger® or 6% PAGE-PLUS™ gels.
Gel pre-run was not long enough. Pre-run the gel until the gel
temperature is 50°C.
Electrophoresis temperature was too high. Run gel at lower
temperature (40–50°C).
(n–1) bands presentFollowing amplification, lengthen the final extension step from
30 minutes at 60°C to 45 minutes. n–1 bands may be generated
when more than 1ng of template DNA is used. This is most
commonly observed with the vWA and D3S1358 amplification
products. Reduce the amount of template DNA used, or reduce
the number of cycles by 2 or 4 cycles (10/20 or 10/18 cycles).
Incorrect matrix patternIncorrect filter set was used or filters in the wrong position for
scanning. Filters for scanning should be:
Position 1: 598
Position 2: 665
Position 3: 505
Position 4: 577
Incorrect colors were assigned to the channels. Channel colors
should be:
Channel 1: red
Channel 2: blue
Channel 3: green
Channel 4: yellow
Extra bands visible in one or all color channelsContamination with another template DNA or previously
amplified DNA. Cross-contamination can be a problem. Use
aerosol-resistant pipette tips, and change gloves regularly.
Artifacts of STR amplification. Amplification of STRs sometimes
generates artifacts that appear as faint peaks one repeat unit
smaller than the allele. Stutter band peak heights will be high if
the samples are overloaded.
Allelic ladder contamination. Store pre- and post-amplification
components separately.
Samples were not completely denatured. Heat-denature samples
at 95°C for 2 minutes, and immediately chill on crushed ice or in
an ice-water bath prior to loading the gel. Do not cool the
samples in a thermal cycler set at 4°C, as this may lead to
artifacts due to DNA re-annealing.
22
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Symptoms
Causes and Comments
BleedthroughIncorrect gray level. Use matrix lanes for color separation.
Confirm that the bands selected are from the appropriate
scanning channel.
Incorrect colors were assigned to the channels. Repeat gray level
adjustment (Figure 2).
Signal was too strong. Use less template in the amplification
reactions, or load less amplified sample onto the gel.
Gel was not run long enough. Bands become more diffuse (less
intense) during the gel run. Gels should be run until the
100-base fragment is near the bottom of the gel.
Imbalance of band intensities across lociToo much template. The system is balanced using 1ng of DNA
template. Using greater than 2ng will lead to overrepresented
smaller alleles and underrepresented larger alleles. Use the
recommended amount of template. Alternatively, reduce the
number of cycles in the amplification program to improve
locus-to-locus balance.
Poor-quality polyacrylamide gel. Prepare acrylamide and buffer
solutions using high-quality reagents. We recommend 5% Long
Ranger® or 6% PAGE-PLUS™ gels.
Too many cycles in the amplification protocol. Use the recommended amplification program; confirm the number of cycles.
Too much enzyme present. Use the recommended amount of
AmpliTaq Gold® DNA polymerase.
Degraded DNA sample. Confirm the DNA integrity by running
an aliquot on an agarose gel. Repurify the template DNA if
necessary.
Miscellaneous balance problems. Thaw the 10X Primer Pair Mix
and Gold STHR 10X Buffer completely, and vortex for 15 seconds
before using. Calibrate thermal cyclers and pipettes routinely.
Decreasing the annealing temperatures by 1–2°C also can
improve the balance.
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7.
Troubleshooting (continued)
Symptoms
Causes and Comments
Imbalance of band intensities across loci
Too much template from card or membrane punches of
(continued)bloodstains. Cards or membranes that bind DNA tightly can
contain more template DNA than recommended. This will lead
to overrepresented smaller alleles and underrepresented larger
alleles. Use the recommended amount of template by using a
smaller punch of the membrane. Alternatively, fewer cycles of
amplification can compensate for this type of unevenness of
product yield (i.e., 10/20 or 10/18 cycling).
Incorrect plates or tubes were used. Use only MicroAmp®
tubes or plates. Due to potential differences in heat transfer
capabilities, plates or tubes from other manufacturers might
result in imbalance.
Poor separation of alleles in ladder lanes or
Gel was not run long enough. Run gel as long as possible
difficulty resolving microvariant alleleswithout running the 100-base ILS fragment off the bottom of the
gel. If necessary run the gel for additional time after the first
scan, then scan the gel a second time to achieve greater
separation of larger alleles.
Scanning resolution was too low. Default scanning resolution is
150dpi. If necessary this resolution can be increased to 300dpi,
which should help sharpen bands.
White background with low signal intensityPart of the white spacers were scanned. Rescan the gel, being
careful not to scan any portion of the spacers, remove the
spacers for scanning or use black electrical tape to cover the
spacers. We recommend using clear spacers.
Dark, grainy background with low signal intensityFocusing point may need to be adjusted. Perform multiple scans
using different focusing points. Adjust focusing point if
necessary.
Plates were improperly washed. Improper washing of plates can
cause a soap residue to build up on the plates, which can cause
background fluorescence. Plate may be soaked in 1N NaOH to
remove residue. Do not use ethanol to clean plates prior to
scanning.
24
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8.References
1.
Edwards, A. et al. (1991) DNA typing with trimeric and tetrameric tandem repeats: Polymorphic loci, detection
systems, and population genetics. In: The Second International Symposium on Human Identification 1991,
Promega Corporation, 31–52.
2.
Edwards, A. et al. (1991) DNA typing and genetic mapping with trimeric and tetrameric tandem repeats.
Am. J. Hum. Genet. 49, 746–56.
3.
Edwards, A. et al. (1992) Genetic variation at five trimeric and tetrameric tandem repeat loci in four human
population groups. Genomics 12, 241–53.
4.
Warne, D. et al. (1991) Tetranucleotide repeat polymorphism at the human β-actin related pseudogene 2
(actbp2) detected using the polymerase chain reaction. Nucl. Acids Res. 19, 6980.
5.
Ausubel, F.M. et al. (1993) Unit 15: The polymerase chain reaction. In: Current Protocols in Molecular Biology,
Vol. 2, Greene Publishing Associates, Inc., and John Wiley and Sons, NY.
6.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Chapter 14: In vitro amplification of DNA by the polymerase
chain reaction. In: Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, New York.
7.
PCR Technology: Principles and Applications for DNA Amplification (1989) ed., Erlich, H.A., Stockton Press,
New York.
8.
PCR Protocols: A Guide to Methods and Applications (1990) eds., Innis, M.A. et al. Academic Press, San Diego.
9.
Presley, L.A. et al. (1992) The implementation of the polymerase chain reaction (PCR) HLA DQ alpha typing
by the FBI laboratory. In: The Third International Symposium on Human Identification 1992, Promega
Corporation, 245–69.
10. Hartmann, J.M. et al. (1991) Guidelines for a quality assurance program for DNA analysis. Crime Laboratory
Digest 18, 44–75.
11. Levinson, G. and Gutman, G.A. (1987) Slipped-strand mispairing: A major mechanism for DNA sequence
evolution. Mol. Biol. Evol. 4, 203–21.
12. Schlotterer, C. and Tautz, D. (1992) Slippage synthesis of simple sequence DNA. Nucl. Acids Res. 20, 211–5.
13. Smith, J.R. et al. (1995) Approach to genotyping errors caused by nontemplated nucleotide addition by Taq
DNA polymerase. Genome Res. 5, 312–7.
14. Magnuson, V.L. et al. (1996) Substrate nucleotide-determined non-templated addition of adenine by Taq DNA
polymerase: Implications for PCR-based genotyping. BioTechniques 21, 700–9.
15. Walsh, P.S., Fildes, N.J. and Reynolds, R. (1996) Sequence analysis and characterization of stutter products at
the tetranucleotide repeat locus vWA. Nucl. Acids Res. 24, 2807–12.
16. Moller, A., Meyer, E. and Brinkmann, B. (1994) Different types of structural variation in STRs: HumFES/FPS,
HumVWA and HumD21S11. Int. J. Leg. Med. 106, 319–23.
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25
8.
References (continued)
17. Brinkmann, B., Moller A. and Wiegand, P. (1995) Structure of new mutations in 2 STR systems. Int. J. Leg. Med.
107, 201–3.
18. Griffiths, R. et al. (1998) New reference allelic ladders to improve allelic designation in a multiplex STR system.
Int. J. Legal Med. 111, 267–72.
19. Bär W. et al. (1997) DNA recommendations: Further report of the DNA Commission of the ISFH regarding the
use of short tandem repeat systems. Int. J. Legal Med. 110, 175–6.
20. Gill, P. et al. (1997) Considerations from the European DNA profiling group (EDNAP) concerning STR nomenclature. Forensic Sci. Int. 87, 185–92.
21. Frégeau, C.J. et al. (1995) Characterization of human lymphoid cell lines GM9947 and GM9948 as intra- and
interlaboratory reference standards for DNA typing. Genomics 28, 184–97.
22. Levadokou, E. et al. (2001) Allele frequencies for fourteen STR loci of the PowerPlex® 1.1 and 2.1 Multiplex
Systems and Penta D Locus in Caucasians, African-Americans, Hispanics and other populations of the United
States of America and Brazil. J. Forensic Sci. 46, 736–61.
23. Lins, A.M. et al. (1998) Development and population study of an eight-locus short tandem repeat (STR) multiplex system. J. Forensic Sci. 43, 1168–80.
24. Puers, C. et al. (1993) Identification of repeat sequence heterogeneity at the polymorphic STR locus
HUMTH01[AATG]n and reassignment of alleles in population analysis using a locus-specific allelic ladder.
Am. J. Human Genet. 53, 953–8.
25. Hammond, H. et al. (1994) Evaluation of 13 short tandem repeat loci for use in personal identification
applications. Am. J. Hum. Genet. 55, 175–89.
26. Bever, R.A. and Creacy, S. (1995) Validation and utilization of commercially available STR multiplexes for
parentage analysis. In: Proceedings from the Fifth International Symposium on Human Identification 1994,
Promega Corporation, 61–8.
27. Sprecher, C.J. et al. (1996) A general approach to analysis of polymorphic short tandem repeat loci.
BioTechniques 20, 266–76.
28. Lins, A.M. et al. (1996) Multiplex sets for the amplification of polymorphic short tandem repeat loci—silver stain
and fluorescent detection. BioTechniques 20, 882–9.
29. Jones, D.A. (1972) Blood samples: Probability of discrimination. J. Forensic Sci. Soc. 12, 355–9.
30. Brenner, C. and Morris, J.W. (1990) In: Proceedings from the International Symposium on Human
Identification 1989, Promega Corporation, 21–53.
31. Mandrekar, P.V., Krenke, B.E. and Tereba, A. (2001) DNA IQ™: The intelligent way to purify DNA.
Profiles in DNA 4(3), 16.
32. Krenke, B.E. et al. (2005) Development of a novel, fluorescent, two-primer approach to quantitative PCR.
Profiles in DNA 8(1), 3–5.
Additional STR references can be found at: www.promega.com/geneticidentity/
26
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9.Appendix
9.A. Advantages of Using the Loci in the PowerPlex® 16 BIO System
The loci included in the PowerPlex® 16 BIO System (Tables 6, 7 and 8) were selected because they satisfy the needs
of several major standardization bodies throughout the world. For example, the United States Federal Bureau of
Investigation (FBI) has selected 13 STR core loci for typing prior to searching or including (submitting) samples in the
Combined DNA Index System (CODIS), the U.S. national database of convicted offender profiles. The PowerPlex® 16
BIO System amplifies all CODIS core loci in a single reaction.
The PowerPlex® 16 BIO System also contains two low-stutter, highly polymorphic pentanucleotide repeat loci,
Penta E and Penta D. These additional loci add significantly to the discrimination power of the system, making the
PowerPlex® 16 BIO System a single-amplification system with a power of exclusion sufficient to resolve paternity
disputes definitively. In addition, the extremely low stutter seen with Penta E and Penta D makes them ideal loci to
evaluate DNA mixtures often encountered in forensic casework. Finally, the Amelogenin locus is included in the
PowerPlex® 16 BIO System to allow gender identification of each sample. Table 8 lists the system alleles revealed in
commonly available standard DNA templates.
We have carefully selected STR loci and primers to avoid or minimize artifacts, including those associated with Taq
DNA polymerase, such as repeat slippage and terminal nucleotide addition. Repeat slippage (11,12), sometimes called
“n–4 bands,” “stutter” or “shadow bands,” is due to the loss of a repeat unit during DNA amplification, somatic
variation within the DNA, or both. The amount of this artifact observed depends primarily on the locus and DNA
sequence being amplified.
Terminal nucleotide addition (13,14) occurs when Taq DNA polymerase adds a nucleotide, generally adenine, to the
3´ ends of amplified DNA fragments in a template-independent manner. The efficiency with which this occurs varies
with different primer sequences. Thus, an artifact band one base shorter than expected (i.e., missing the terminal
addition) is sometimes seen. We have modified primer sequences and added a final extension step of 60°C for
30 minutes (15) to the amplification protocol to provide conditions for essentially complete terminal nucleotide
addition when recommended amounts of DNA template are used.
The presence of microvariant alleles (alleles differing from one another by lengths other than the repeat length)
complicates interpretation and assignment of alleles. There appears to be a correlation between a high degree of
polymorphism, a tendency for microvariants and increased mutation rate (16,17). Thus, FGA and D21S11 display
numerous, relatively common microvariants. For reasons yet unknown, the highly polymorphic Penta E locus does
not display frequent microvariants (Table 7).
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27
Table 6. The PowerPlex® 16 BIO System Locus-Specific Information.
GenBank® Locus and
Locus Definition
Repeat Sequence1
5´→ 3´
Label
Chromosomal
Location
Penta E
FL
15q
D18S51
FL
18q21.3
D21S11
FL
21q11–21q21
TH01
FL
11p15.5
D3S1358
FL
3p
FGA
Rhodamine Red™-X
4q28
HUMFIBRA, Human
fibrinogen alpha chain gene
TTTC Complex (18)
TPOX
Rhodamine Red™-X
2p24–2pter
HUMTPOX, Human thyroid
peroxidase gene
AATG
D8S1179
Rhodamine Red™-X
8q
vWA
Rhodamine Red™-X
12p12–pter
Amelogenin2
Rhodamine Red™-X
Xp22.1–22.3 and Y
Penta D
JOE
21q
CSF1PO
JOE
5q33.3–34
STR Locus
NA
AAAGA
HUMUT574
HUMD21LOC
AGAA (18)
TCTA Complex (18)
HUMTH01, Human tyrosine
hydroxylase gene
NA
AATG (18)
TCTA Complex
NA
TCTA Complex (18)
HUMVWFA31, Human von
Willebrand factor gene
TCTA Complex (18)
HUMAMEL, Human Y
chromosomal gene for
Amelogenin-like protein
NA
NA
AAAGA
HUMCSF1PO, Human c-fms
proto-oncogene for CSF-1
receptor gene
AGAT
D16S539
JOE
16q24–qter
NA
GATA
D7S820
JOE
7q11.21–22
NA
GATA
D13S317
JOE
13q22–q31
NA
TATC
D5S818
JOE
5q23.3–32
NA
AGAT
1
The August 1997 report (19,20) of the DNA Commission of the International Society for Forensic Haemogenetics
(ISFH) states, “1) for STR loci within coding genes, the coding strand shall be used and the repeat sequence motif
defined using the first possible 5´ nucleotide of a repeat motif; and 2) for STR loci not associated with a coding gene,
the first database entry or original literature description shall be used”.
Amelogenin is not an STR but displays a 106-base, X-specific band and a 112-base, Y-specific band.
2
FL = fluorescein.
JOE = 6-carboxy-4´,5´-dichloro-2´,7´-dimethoxyfluorescein.
NA = not applicable.
28
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Table 7. The PowerPlex® 16 BIO System Allelic Ladder Information.
Label
Size Range of
Allelic Ladder
Components1,2
(bases)
Penta E
FL
379–474
5–24
D18S51
FL
290–366
8–10, 10.2, 11–13, 13.2, 14–27
D21S11
FL
203–259
24, 24.2, 25, 25.2, 26–28, 28.2,
29, 29.2, 30, 30.2, 31, 31.2, 32,
32.2, 33, 33.2, 34, 34.2, 35, 35.2,
36–38
TH01
FL
156–195
4–9, 9.3, 10–11, 13.3
D3S1358
FL
115–147
12–20
FGA
Rhodamine Red™-X
322–444
16–30, 31.2, 43.2, 44.2, 45.2, 46.2
TPOX
Rhodamine Red™-X
262–290
6–13
D8S1179
Rhodamine Red™-X
203–247
7–18
Rhodamine Red™-X
123–171
10–22
STR Locus
vWA
Amelogenin
Repeat Numbers of Allelic
Ladder Components
Rhodamine Red™-X
106, 112
X, Y
Penta D
JOE
376–449
2.2, 3.2, 5, 7–17
CSF1PO
JOE
321–357
6–15
D16S539
JOE
264–304
5, 8–15
D7S820
JOE
215–247
6–14
D13S317
JOE
176-208
7–15
D5S818
JOE
119–155
7–16
5
Repeat Numbers
of Alleles Not
Present in
Allelic Ladder3,4
20.3
18.2, 19.2, 22.2,
23.2, 24.2, 25.2
Lengths of each allele in the allelic ladders have been confirmed by sequence analyses.
1
When using an internal lane standard such as the Internal Lane Standard 600 BIO, calculated sizes of allelic ladder
components may differ from those listed. This occurs because different sequences in allelic ladder and ILS components
may cause differences in migration. The dye label also affects migration of alleles.
2
The alleles listed are those with a frequency of >1/1000.
3
For a current list of microvariants, see the Variant Allele Report published at the U.S. National Institute of Standards
and Technology (NIST) web site at: www.cstl.nist.gov/div831/strbase/
4
Amelogenin is not an STR but displays a 106-base, X-specific band and a 112-base, Y-specific band.
5
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29
Table 8. The PowerPlex® 16 BIO System Allele Determinations in Commonly Available Standard DNA
Templates.
Standard DNA Templates1
STR Locus
K562
9947A
99483
2
2800M
Penta E
5, 14
12, 13
11, 11
7, 14
D18S51
15, 16
15, 19
15, 18
16, 18
D21S11
29, 30, 31
30, 30
29, 30
29, 31.2
9.3, 9.3
8, 9.3
6, 9.3
6, 9.3
TH01
D3S1358
16, 16
14, 15
15, 17
17, 18
FGA
21, 24
23, 24
24, 26
20, 23
8, 9
8, 8
8, 9
11, 11
TPOX
D8S1179
12, 12
13, 13
12, 13
14, 15
vWA
16, 16
17, 18
17, 17
16, 19
X, X
X, X
X, Y
X, Y
Amelogenin
Penta D
9, 13
12, 12
8, 12
12, 13
CSF1PO
9, 10
10, 12
10, 11, 12
12, 12
D16S539
11, 12
11, 12
11, 11
9, 13
D7S820
9, 11
10, 11
11, 11
8, 11
D13S317
8, 8
11, 11
11, 11
9, 11
D5S818
11, 12
11, 11
11, 13
12, 12
Information on strains K562, 9947A and 9948 is available online at: http://ccr.coriell.org
Strain K562 is available from the American Type Culture Collection: www.atcc.org (Manassas, VA).
Information about the use of 9947A and 9948 DNA as standard DNA templates can be found in reference 21.
1
Strain K562 displays three alleles at the D21S11 locus.
2
Strain 9948 displays three alleles at the CSF1PO locus. The peak height for allele 12 is much lower than those for
alleles 10 and 11.
3
30
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9.B. Power of Discrimination
The fifteen STR loci amplified with the PowerPlex® 16 BIO System provide powerful discrimination. Population
statistics for these loci and their various multiplex combinations are displayed in Table 9. These data were developed as
part of a collaboration (22) with The Bode Technology Group (Springfield, VA), North Carolina Bureau of Investigation
(Raleigh, NC), Palm Beach County Sheriff’s Office (West Palm Beach, FL), Virginia Division of Forensic Science
(Richmond, VA) and Charlotte/ Mecklenburg Police Department Laboratory (NC). Generation of these data includes
analysis of over 200 individuals from African-American, Caucasian-American and Hispanic-American populations.
Data for Asian-Americans includes analysis of over 150 individuals. For additional population data for STR loci, see
references 23–28 and the Short Tandem Repeat DNA Internet DataBase at: www.cstl.nist.gov/div831/strbase/
Table 9 shows the matching probability (29) for the PowerPlex® 16 BIO System in various populations. The matching
probability of this system ranges from 1 in 1.83 × 1017 for Caucasian-Americans to 1 in 1.41 × 1018 for African-Americans.
A measure of discrimination often used in paternity analyses is the paternity index (PI), a means for presenting the
genetic odds in favor of paternity given the genotypes for the mother, child and alleged father. The typical paternity
indices for the PowerPlex® 16 BIO System are shown in Table 9. This system provides typical paternity indices
exceeding 500,000 in each population group. An alternative calculation used in paternity analyses is the power of
exclusion (30). This value, calculated for the system, exceeds 0.999998 in all populations tested (Table 9).
Table 9. Matching Probabilities, Paternity Indices and Power of Exclusion of the PowerPlex® 16 BIO
System in Various Populations.
African-American Caucasian-American Hispanic-American Asian-American
1 in 1.41 × 1018
1 in 1.83 × 1017
1 in 2.93 × 1017
1 in 3.74 × 1017
Paternity Index
2,510,000
1,520,000
522,000
4,110,000
Power of Exclusion
0.9999996
0.9999994
0.9999983
0.9999998
Matching Probability
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31
9.C. DNA Extraction and Quantitation Methods and Automation Support
The DNA IQ™ System (Cat.# DC6700) is a DNA isolation system designed specifically for forensic and paternity
samples (31). This novel system uses paramagnetic particles to prepare clean samples for STR analysis easily and
efficiently and can be used to extract DNA from stains or liquid samples, such as blood or solutions. The DNA IQ™
Resin eliminates PCR inhibitors and contaminants frequently encountered in casework samples. With larger samples,
the DNA IQ™ System delivers a consistent amount of total DNA. The system has been used to isolate and quantify
DNA from routine sample types including buccal swabs, stains on FTA® paper and liquid blood. Additionally, DNA
has been isolated from casework samples such as tissue, differentially separated sexual assault samples and stains on
support materials. The DNA IQ™ System has been tested with the PowerPlex® Systems to ensure a streamlined
process. See Section 9.G for ordering information.
For applications requiring human-specific DNA quantification, the Plexor® HY System (Cat.# DC1001, DC1000) has
been developed (32). See Section 9.G for ordering information.
For information about automation of Promega chemistries on automated workstations using Identity Automation™
solutions, contact your local Promega Branch Office or Distributor (contact information available at:
www.promega.com/support/worldwide-contacts/), e-mail: [email protected] or visit:
www.promega.com/idautomation/
9.D. The Internal Lane Standard 600 BIO
The Internal Lane Standard (ILS) 600 BIO contains 21 DNA fragments of 80, 100, 120, 140, 160, 180, 200, 225,
250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550 and 600 bases in length. Each fragment is labeled with
Texas Red®-X and may be detected separately (as a fourth color) in the presence of PowerPlex® 16 BIO amplified
material using the Hitachi FMBIO® II Fluorescence Imaging System. The ILS 600 BIO is designed for use in each gel
lane to increase precision in analyses when using the PowerPlex® 16 BIO System. A protocol for preparation and use of
this internal lane standard is provided in Section 5.C.
32
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9.E. Preparing the PowerPlex® 16 BIO System PCR Amplification Mix
Use Table 10 to calculate the required amount of each component of the PCR amplification mix. Multiply the volume
(µl) per sample by the total number of reactions to obtain the final PCR amplification mix volume (µl).
Table 10. PCR Amplification Mix for the PowerPlex® 16 BIO System.
PCR Master
Mix Component
Gold STHR 10X Buffer
PowerPlex 16 BIO 10X Primer Pair Mix
®
AmpliTaq Gold® DNA polymerase1
nuclease-free water
2
Volume
Per Sample
×
Number of
Reactions
2.5µl
×
=
=
2.5µl
×
=
0.8µl (4u)
×
=
µl
×
=
×
=
up to 19.2µl
×
=
25µl
×
=
Final Volume
(µl)
Per tube
template DNA volume2 (0.5–1ng)
total reaction volume
Assumes the AmpliTaq Gold® DNA polymerase is at 5u/µl. If the enzyme concentration is different, the volume of
enzyme used must be adjusted accordingly.
1
The PCR amplification mix volume added to the template DNA volume should total 25µl. Consider the volume of
template DNA, and add nuclease-free water to the PCR amplification mix to bring the final volume of the final reaction
to 25µl.
2
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33
9.F. Composition of Buffers and Solutions
10% ammonium persulfate
Add 0.05g of ammonium persulfate to 500µl of deionized
water.
Bromophenol Blue Loading Solution
10mMNaOH
95%formamide
0.05% bromophenol blue
Gel Tracking Dye
10mMNaOH
95%formamide
0.05% bromophenol blue
0.05% xylene cyanol FF
Gold STHR 10X Buffer
500mM KCl
100mM Tris-HCl (pH 8.3 at 25°C)
15mMMgCl2
1%Triton® X-100
2mM each dNTP
1.6mg/ml BSA
34
TBE 10X buffer
107.8g Tris base
7.44g EDTA (Na2EDTA • 2H2O)
~55.0g boric acid
Dissolve the Tris base and EDTA in 800ml of deionized
water. Slowly add the boric acid, and monitor the pH
until the desired pH of 8.3 is obtained. Bring the volume
to 1 liter with deionized water.
TE–4 buffer [10mM Tris-HCl, 0.1mM EDTA (pH
8.0)]
1.21g Tris base
0.037g EDTA (Na2EDTA • 2H2O)
Dissolve Tris base and EDTA in 900ml of deionized
water. Adjust to pH 8.0 with HCl. Bring the final volume
to 1 liter with deionized water.
TE–4 buffer with 20µg/ml glycogen
1.21g Tris base
0.037g EDTA (Na2EDTA • 2H2O)
20µg/ml glycogen
Dissolve Tris base and EDTA in 900ml of deionized
water. Adjust to pH 8.0 with HCl. Add glycogen. Bring
the final volume to 1 liter with deionized water.
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9.G. Related Products
Product
SizeCat.#
PowerPlex® 16 Monoplex System, Penta E (Fluorescein)
100 reactions
DC6591
PowerPlex 16 Monoplex System, Penta D (JOE)
100 reactions
DC6651
®
Not For Medical Diagnostic Use.
Accessory Components
SizeCat.#
Product
2800M Control DNA*
25µl
DD7101
500µlDD7251
Gold STHR 10X Buffer
1.2ml
DM2411
Nuclease-Free Water
50ml
P1193
*Not For Medical Diagnostic Use.
Sample Preparation Systems
SizeCat.#
Product
DNA IQ™ System
100 reactions
DC6701
400 reactions
DC6700
50 samples
DC6801
200 samples
DC6800
1 each
AS3060
DNA IQ™ Reference Sample Kit for Maxwell 16
48 preps
AS1040
DNA IQ™ Casework Pro Kit for Maxwell® 16*
48 preps
AS1240
Plexor HY System*
200 reactions
DC1001
800 reactions
DC1000
10 pack
V1391
Differex™ System*
Maxwell® 16 Forensic Instrument*
®
®
Slicprep™ 96 Device
*Not for Medical Diagnostic Use.
Polyacrylamide Gel Electrophoresis Reagents
Product
Ammonium Persulfate, Molecular Grade
TBE Buffer, 10X, Molecular Biology Grade
Urea
SizeCat.#
25g
V3131
1L
V4251
1kgV3171
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35
9.H. Summary of Changes
The following changes were made to the 7/15 revision of this document:
1.
The patent/license statements were updated.
2.
The discontinued 100-reaction size of the product was removed.
3.
The document design was updated.
PowerPlex® 16 BIO System incorporates dye conjugates made with the Rhodamine Red™-X and Texas Red®-X fluorescent reactive dyes, which are
licensed from Molecular Probes, Inc., under U.S. Pat. Nos. 5,798,276 and 5,846,737 for DNA analysis. Rhodamine Red is a trademark of Molecular
Probes, Inc., and Texas Red is a registered trademark of Molecular Probes, Inc.
(a)
(b)
U.S. Pat. No. 6,238,863, Chinese Pat. No. ZL99802696.4, European Pat. No. 1058727, Japanese Pat. No. 4494630 and other patents pending.
Australian Pat. No. 724531, Canadian Pat. No. 2,251,793, Korean Pat. No. 290332, Singapore Pat. No. 57050, Japanese Pat. Nos. 3602142 and
4034293, Chinese Pat. Nos. ZL99813729.4 and ZL97194967.0, European Pat. No. 0960207 and other patents pending.
(c)
(d)
The purchase of this product does not convey a license to use AmpliTaq Gold® DNA polymerase. You should purchase AmpliTaq Gold® DNA polymerase
licensed for the forensic and human identity field directly from your authorized enzyme supplier.
Allele sequences for one or more of the loci vWA, FGA, D8S1179, D21S11 and D18S51 in allelic ladder mixtures is licensed under U.S. Pat. Nos.
7,087,380, 7,645,580, Australia Pat. No. 2003200444 and corresponding patent claims outside the US.
(e)
© 2001, 2002, 2007, 2008, 2011, 2012, 2015 Promega Corporation. All Rights Reserved.
Maxwell, Plexor and PowerPlex are registered trademarks of Promega Corporation. Differex, DNA IQ and Slicprep are trademarks of Promega
Corporation.
AmpliTaq Gold and GeneAmp are registered trademarks of Roche Molecular Systems, Inc. ART is a registered trademark of Molecular Bio-Products, Inc.
FMBIO is a registered trademark of Hitachi Software Engineering Company, Ltd. FTA is a registered trademark of Flinders Technologies, Pty, Ltd., and is
licensed to Whatman. GenBank is a registered trademark of the U.S. Dept. of Health and Human Services. Kimwipes is a registered trademark of
Kimberly-Clark Corporation. Liqui-Nox is a registered trademark of Alconox, Inc. Long Ranger and Long Ranger Singel are registered trademarks of
Cambrex Corporation. Macintosh is a registered trademark of Apple Computer, Inc. MicroAmp is a registered trademark of Applera Corporation.
Nalgene is a registered trademark of Nalge Nunc International. PAGE-PLUS is a trademark of Amresco, Inc. Rhodamine Red is a trademark of Molecular
Probes, Inc. Texas Red is a registered trademark of Molecular Probes, Inc. Triton is a registered trademark of Union Carbide Chemicals & Plastics
Technology Corporation.
Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for more information.
All prices and specifications are subject to change without prior notice.
Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the most up-to-date
information on Promega products.
36
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