Promega PowerPlex ESI 17 Fast System Manuel utilisateur

TECHNICAL MANUAL

PowerPlex®ESI 17 Fast System

for Use on the

Applied Biosystems®

Genetic Analyzers

Instructions for Use of Products

DC1720 and DC1721

Revised 10/23

TMD041

PowerPlex®ESI 17 Fast System for Use on

the Applied Biosystems®Genetic Analyzers

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]

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1. Description............................................................................................................................................3

2. Product Components and Storage Conditions..............................................................................................4

3. Before You Begin....................................................................................................................................5

3.A. Precautions...................................................................................................................................5

3.B. Spectral Calibration........................................................................................................................6

 4. ProtocolsforDNAAmplicationUsingthePowerPlex®ESI 17 Fast System......................................................6

4.A. AmplicationofExtractedDNAina25µlReactionVolume....................................................................7

4.A. AmplicationofExtractedDNAina25µlReactionVolume....................................................................8

4.B. DirectAmplicationofDNAfromStorageCardPunchesina25µlReactionVolume................................ 10

4.C. DirectAmplicationofDNAfromSwabsina25µlReactionVolume ..................................................... 14

5. Instrument Setup and Sample Preparation ................................................................................................ 17

5.A. DetectionofAmpliedFragmentsUsingtheAppliedBiosystems®3500or3500xLGeneticAnalyzer ......... 17

5.B. DetectionofAmpliedFragmentsUsingtheAppliedBiosystems®3130 or 3130xl GeneticAnalyzerwith

DataCollectionSoftware,Version3.0or4.0..................................................................................... 29

6. Data Analysis ....................................................................................................................................... 32

6.A. ImportingPowerPlex®ESIFastPanels,BinsandStutterTextFilesintoGeneMapper®ID-XSoftware ......... 32

6.B. ImportingtheWENILS500ESSSizeStandardintoGeneMapper®ID-XSoftware .................................... 33

6.C. CreatingaSizeStandardwithGeneMapper®ID-XSoftware................................................................. 33

6.D. CreatingaCaseworkAnalysisMethodwithGeneMapper®ID-XSoftware............................................... 35

6.E. CreatingaDatabasingorPaternityAnalysisMethodwithGeneMapper®ID-XSoftware............................ 40

6.F. ImportingPowerPlex®ESIFastPanelsandBinsTextFilesintoGeneMapper®IDSoftware,Version3.2...... 43

6.G. ImportingtheWENILS500ESSSizeStandardintoGeneMapper®IDSoftware,Version3.2...................... 44

6.H. CreatingaSizeStandardwithGeneMapper®IDSoftware,Version3.2................................................... 45

6.I. CreatingaCaseworkAnalysisMethodwithGeneMapper®IDSoftware,Version3.2 ................................ 47

6.J. CreatingaDatabasingorPaternityAnalysisMethodwithGeneMapper®ID Software,Version3.2.............. 51

6.K. Controls ..................................................................................................................................... 54

6.L. Results....................................................................................................................................... 54

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 7. Troubleshooting ................................................................................................................................... 58

7.A. AmplicationandFragmentDetection ............................................................................................. 58

7.B. AmplicationofExtractedDNA ...................................................................................................... 61

7.C. DirectAmplicationofDNAfromStorageCardPunches..................................................................... 63

7.D. DirectAmplicationofDNAfromSwabs .......................................................................................... 67

7.E. GeneMapper®ID-XSoftware .......................................................................................................... 70

7.F. GeneMapper®IDSoftware ............................................................................................................. 72

 8. References .......................................................................................................................................... 76

 9. Appendix............................................................................................................................................. 78

9.A. AdvantagesofUsingtheLociinthePowerPlex®ESI 17 Fast System .................................................... 78

9.B. TheWENInternalLaneStandard500ESS......................................................................................... 82

9.C. DirectAmplicationofDNAfromStorageCardPunchesina12.5µlReactionVolume ............................. 82

9.D. DirectAmplicationofDNAfromSwabsina12.5µlReactionVolume................................................... 87

9.E. Composition of Buffers and Solutions ............................................................................................. 90

9.F. RelatedProducts.......................................................................................................................... 91

9.G. SummaryofChanges.................................................................................................................... 91

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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 (5–9). Alleles of STR loci are differentiated by the

number of copies of the repeat sequence contained within the amplied region and are distinguished from one

another using uorescence detection following electrophoretic separation.

The PowerPlex®ESI 17 Fast System(a,b) is used for human identication applications including forensic analysis,

relationship testing and research use. This system allows co-amplication and four-color uorescent detection of

seventeen loci (sixteen STR loci and Amelogenin), including D22S1045, D2S1338, D19S433, D3S1358, Amelogenin,

D2S441, D10S1248, D1S1656, D18S51, D16S539, D12S391, D21S11, vWA, TH01, SE33, FGA and D8S1179.

The PowerPlex®ESI 17 Fast System is designed with six of the original seven European Standard Set (ESS) loci

(D3S1358, D18S51, TH01, vWA, D8S1179 and the more common FGA alleles) along with D16S539 and D19S433 as

smaller amplicons (<250bp), while the loci recommended by the European Network of Forensic Science Institutes

(ENFSI) and European DNA Proling Group (EDNAP) (D1S1656, D2S441, D10S1248, D12S391 and D22S1045) are

present as larger amplicons. A complementary system, the PowerPlex® ESX 17 Fast System, amplies the same

seventeen loci present in the PowerPlex®ESI 17 Fast System but with the new ENFSI/EDNAP loci designed as

mini-STRs (<125bp; D2S441, D10S1248 and D22S1045) or midi-STRs (125–185bp; D1S1656 and D12S391).

Therefore, these two STR systems can be used to complement each other when analyzing degraded or challenging

samples to maximize recovery of allelic information from as many loci as possible and allow conrmation of results

obtained with the other system.

The PowerPlex®ESI 17 Fast System and all system components are manufactured in accordance with ISO

18385:2016. All necessary materials are provided to amplify STR regions of human genomic DNA, including a

hot-start thermostable DNA polymerase, which is a component of the PowerPlex®ESI/ESX Fast 5X Master Mix.

This manual contains a protocol for use of the PowerPlex®ESI 17 Fast System with the GeneAmp®PCR System

9700, Veriti®96-Well Thermal Cycler and ProFlex® PCR System in addition to protocols to separate amplied

products and detect separated material on the Applied Biosystems®3130, 3130xl, 3500 and 3500xL Genetic

Analyzers. Protocols to operate the uorescence-detection instruments should be obtained from the instrument

manufacturer. Amplication and detection instrumentation may vary. You may need to optimize protocols including

amount of template DNA, cycle number, injection conditions and loading volume for your laboratory instrumentation.

In-house validation should be performed.

Information about other Promega uorescent STR systems is available upon request from Promega or online at:

www.promega.com

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2. Product Components and Storage Conditions

PRODUCT SIZE CAT.#

PowerPlex®ESI 17 Fast System 100 reactions DC1721

Not For Medical Diagnostic Use. This system contains sufcient reagents for 100 reactions of 25µl each. Includes:

Pre-amplicationComponentsBox

•500µl PowerPlex®ESI/ESX Fast 5X Master Mix

•250µl PowerPlex®ESI 17 Fast 10X Primer Pair Mix

• 25µl 2800M Control DNA, 10ng/µl

• 5×1,250µl Water,AmplicationGrade

Post-amplicationComponentsBox

•50µl PowerPlex®ESI17FastAllelicLadderMix

• 200µl WENInternalLaneStandard500ESS

PRODUCT SIZE CAT.#

PowerPlex®ESI 17 Fast System 400 reactions DC1720

Not For Medical Diagnostic Use. This system contains sufcient reagents for 400reactions of 25µl each. Includes:

Pre-amplicationComponentsBox

• 4 × 500µl PowerPlex®ESI/ESX Fast 5X Master Mix

• 4 × 250µl PowerPlex®ESI 17 Fast 10X Primer Pair Mix

• 25µl 2800M Control DNA, 10ng/µl

•10×1,250µl Water,AmplicationGrade

Post-amplicationComponentsBox

• 4 × 50µl PowerPlex®ESI17FastAllelicLadderMix

• 2×200µl WENInternalLaneStandard500ESS

The PowerPlex®ESI 17 Fast Allelic Ladder Mix is provided in a separate, sealed bag for shipping. This component

should be moved to the post-amplication box after opening. The Water, Amplication Grade, is provided in a

separate, sealed bag for shipping. This component should be moved to the pre-amplication box after opening.

Storage Conditions: Upon receipt, store all components at –30°C to –10°C in a nonfrost-free freezer. Make sure that

the 2800M Control DNA is stored at +2°C to +10°C for atleast24hours before use; do not refreeze. After the rst

use, store the WEN Internal Lane Standard (WEN ILS) 500 ESS at +2°C to +10°C, protected from light; do not refreeze.

The PowerPlex®ESI 17 Fast 10X Primer Pair Mix, PowerPlex®ESI 17 Fast Allelic Ladder Mix and WEN ILS 500 ESS

are light-sensitive and must be stored in the dark. We strongly recommend that pre-amplication and post-ampli-

cation reagents be stored and used separately with different pipettes, tube racks, etc.

Optional: The PowerPlex®ESI 17 Fast System components may be stored for up to 1 year at +2°C to +10°C without

loss of activity.

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Available Separately

PRODUCT SIZE CAT.#

PunchSolution™ Kit 100 preps DC9271

SwabSolution™ Kit 100 preps DC8271

5X AmpSolution™ Reagent 500µl DM1231

The PunchSolution™ Kit is required to process nonlytic storage card punches prior to direct amplication. The

SwabSolution™ Kit is required to process swabs prior to direct amplication. The 5X AmpSolution™ Reagent is

required for direct amplication of DNA from storage card punches and swab extracts. Both the PunchSolution™ Kit

and SwabSolution™ Kit contain the 5X AmpSolution™ Reagent.

The proper panels, bins and stutter text les and size standard .xml les for use with GeneMapper®ID and ID-X

software can be downloaded at: www.promega.com/resources/software-rmware/str-analysis/genemapper-id-

software-panels-and-bin-sets/

Matrix standards are required for initial setup of the color separation matrix. Matrix standards are provided

separately and are available for the Applied Biosystems®3130, 3130xl, 3500 and 3500xL Genetic Analyzers

(PowerPlex® 5C Matrix Standard, Cat.# DG4850).

3. BeforeYouBegin

3.A. Precautions

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 (10,11). Guidelines for the validation process are published in the

Internal Validation Guide of Autosomal STR Systems for Forensic Laboratories (12).

The quality of puried DNA or direct-amplication samples, small changes in buffers, ionic strength, primer

concentrations, reaction volume, choice of thermal cycler and thermal cycling conditions can affect PCR success.

We suggest strict adherence to recommended procedures for amplication and uorescence detection. Additional

research and validation are required if any modications to the recommended protocols are made.

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 template DNA, handling primer pairs, assembling amplication

reactions and analyzing amplication products. Reagents and materials used prior to amplication (Master Mix,

Primer Pair Mix, 2800M Control DNA and Water, Amplication Grade) are provided in a separate box and should be

stored separately from those used following amplication (Allelic Ladder Mix and Internal Lane Standard). 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.

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3.A. Precautions (continued)

Some reagents used in the analysis of STR products are potentially hazardous and should be handled accordingly.

Formamide is an irritant and a teratogen; avoid inhalation and contact with skin. Read the warning label, and take

appropriate precautions when handling this substance. Always wear gloves and safety glasses when working with

formamide.

3.B. Spectral Calibration

Proper spectral calibration is critical to evaluate multicolor systems with the Applied Biosystems®3130, 3130xl,

3500 and 3500xL Genetic Analyzers. A matrix must be generated for each individual instrument.

The PowerPlex®5C Matrix Standard (Cat.# DG4850) is required for spectral calibration on the Applied Biosystems®

3130, 3130xl, 3500 and 3500xL Genetic Analyzers.

For protocols and additional information about spectral calibration, see the PowerPlex®5C Matrix Standard Technical

Manual #TMD049. This manual is available online at: www.promega.com/protocols/

4. ProtocolsforDNAAmplicationUsingthePowerPlex®ESI 17 Fast System

The PowerPlex® ESI 17 Fast System was developed for amplication of extracted DNA and direct-amplication

samples. Slight protocol variations are recommended for optimal performance with each template source. Protocols

for amplication in a 25µl reaction volume using extracted DNA (Section 4.A), lytic and nonlytic storage card

punches (Section4.B) and swabs (Section 4.C) are included in the following amplication sections. Protocols for

amplication in a 12.5µl reaction volume using lytic and nonlytic storage card punches and swabs are included in

Sections 9.C and 9.D, respectively.

The PowerPlex®ESI 17 Fast System is compatible with the GeneAmp®PCR System 9700 thermal cycler with a

gold-plated silver or silver sample block, the Veriti®96-Well Thermal Cycler and the ProFlex®PCR System.

Note: It may be possible to use thermal cyclers other than those listed in this technical manual. Use of thermal

cyclers not listed here may require optimization of cycling conditions and validation in your laboratory. Use of

thermal cyclers with an aluminum block is not recommended with the PowerPlex®ESI 17 Fast System.

The use of gloves and aerosol-resistant pipette tips is highly recommended to prevent cross-contamination. Keep all

pre-amplication and post-amplication reagents in separate rooms. Prepare amplication reactions in a room

dedicated for reaction setup. Use equipment and supplies dedicated for amplication setup.

Meticulous care must be taken to ensure successful amplication. A guide to amplication troubleshooting is

provided in Section 7.

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4.A. AmplicationofExtractedDNAina25µlReactionVolume

The PowerPlex®ESI 17 Fast System is optimized and balanced for 0.5ng of DNA template. The amount of DNA

template used in your laboratory should be based on the results of your internal validation and may be different.

Testing at Promega shows that 30 cycles works well for 0.5ng of puried DNA templates. Developmental validation

of the system showed routine generation of full proles using 30 cycles of amplication with lower amounts of DNA

template down to 62.5pg. Partial proles were typically observed for DNA template amounts of 32pg and below (13).

In-house optimization and validation should be performed to establish the performance of the system in your

laboratory (12).

MaterialstoBeSuppliedbytheUser

•GeneAmp®PCR System 9700 with a gold-plated silver or silver sample block, Veriti®96-Well Thermal Cycler or

ProFlex®PCR System (Applied Biosystems)

• centrifuge compatible with 96-well plates or reaction tubes

•MicroAmp®optical 96-well reaction plates or 0.2ml MicroAmp®reaction tubes (Applied Biosystems)

• aerosol-resistant pipette tips

AmplicationSetup

1. Thaw the PowerPlex®ESI/ESX Fast 5X Master Mix, PowerPlex®ESI 17 Fast 10X Primer Pair Mix and Water,

Amplication Grade, completely.

Note: Centrifuge tubes briey to bring contents to the bottom, and then vortex reagents for 15 seconds before

each use. Do not centrifuge the 10X Primer Pair Mix or 5X Master Mix after vortexing, as this may cause the

reagents to be concentrated at the bottom of the tube.

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 amplication mix for all samples. It

also ensures that each reaction contains the same PCR amplication mix.

3. Use a clean plate for reaction assembly, and label it appropriately. Alternatively, determine the number of

clean, 0.2ml reaction tubes required, and label them appropriately.

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4.A. AmplicationofExtractedDNAina25µlReactionVolume

4. Add the nal volume of each reagent listed in Table1 to a clean tube.

Table1.PCRAmplicationMixforAmplicationofExtractedDNA.

PCRAmplicationMixComponent1

Volume

PerReaction ×Number of

Reactions =Final

Volume

Water, Amplication Grade

to a nal volume

of 25.0µl × =

PowerPlex®ESI/ESX Fast 5X Master Mix 5.0µl × =

PowerPlex®ESI 17 Fast 10X Primer Pair Mix 2.5µl × =

template DNA (0.5ng)2,3,4 up to 17.5µl

total reaction volume 25.0µl

1Add Water, Amplication Grade, to the tube rst, and then add PowerPlex®ESI/ESX Fast 5X Master Mix and PowerPlex®

ESI 17 Fast 10X Primer Pair Mix. The template DNA will be added at Step6.

2Store DNA templates in TE–4 buffer (10mM Tris-HCl [pH 8.0], 0.1mM EDTA) or TE–4 buffer with 20µg/ml glycogen. 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 nal reaction volume. PCR amplication efciency 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.

3Apparent DNA concentrations can differ, depending on the DNA quantication method used (14). We strongly recommend

that you perform experiments to determine the optimal DNA amount based on your DNA quantication method and internal

validation.

4The PowerPlex®ESI 17 Fast System is optimized and balanced for 0.5ng of DNA template. The amount of DNA template

used in your laboratory should be based on the results of your internal validation and may be different.

5. Vortex the PCR amplication mix for 5–10seconds, and then pipet PCR amplication mix into each reaction

well.

Failure to vortex the PCR amplication mix sufciently can result in poor amplication or locus-to-locus

imbalance.

Note: Do not store the PCR amplication mix for a prolonged period. Add the mix to the wells of the reaction

plate as soon as the mix is prepared. Add DNA as soon as possible to each well and follow immediately by

thermal cycling.

6. Add template DNA for each sample to the respective well containing PCR amplication mix.

Note: The PowerPlex®ESI 17 Fast System is optimized and balanced for 0.5ng of DNA template. The amount

of DNA template used in your laboratory should be based on the results of your internal validation and may be

different.

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7. For the positive amplication control, vortex the tube of 2800M Control DNA, and then dilute an aliquot

to 0.5ng in the desired template volume. Add 0.5ng of diluted DNA to a reaction well containing PCR

amplication mix.

8. For the negative amplication control, pipet Water, Amplication Grade, or TE–4 buffer instead of template DNA

into a reaction well containing PCR amplication mix.

9. Seal or cap the plate, or close the tubes.

Optional: Briey centrifuge the plate to bring contents to the bottom of the wells and remove any air bubbles.

ThermalCycling

Amplication and detection instrumentation may vary. You may need to optimize protocols including the amount of

template DNA, cycle number, injection conditions and loading volume for your laboratory instrumentation. Testing at

Promega shows that 30cycles work well for 0.5ng of puried DNA templates. In-house validation should be

performed.

1. Place the reaction plate or tubes in the thermal cycler.

2. Select and run the recommended protocol below.

Notes:

a. When using the ProFlex®PCR System, use the 9700 Simulation Mode as the ramp speed.

b. When using the Veriti®96-Well Thermal Cycler, set the ramping rate to 100%.

c. When using the GeneAmp®PCR System 9700, the program must be run with Max Mode as the ramp

speed. This requires a gold-plated silver or silver sample block. The ramp speed is set after the thermal

cycling run is started. When the ‘Select Method Options’ screen appears, select Max for the ramp speed

and enter the reaction volume.

ThermalCyclingProtocol

96°C for 1 minute, then:

96°C for 5 seconds

60°C for 35 seconds

72°C for 5 seconds

for 30 cycles, then:

60°C for 2 minutes

4°C soak

3. After completion of the thermal cycling protocol, proceed to fragment analysis or store amplied samples at

–20°C protected from light.

Note: Long-term storage of amplied samples at 4°C or higher may produce artifacts.

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