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Promega Corporation

Implementation of Promega STR Amplification Systems for Nonforensic Applications

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Publication Date: 2012

Introduction

Commercial short tandem repeat (STR) kits are valuable tools for determining human genetic profiles in the forensic and paternity testing communities. Because this technology typically produces unique DNA fingerprints for individual human DNA samples, it is a powerful technology that is amenable for use in a large number of nonforensic research applications that require genotyping and analysis of single-source and mixed samples. A number of commercial STR kits are available; Table 1 shows the loci amplified by several of these kits. The purpose of this document is to outline the differences and similarities among Promega PowerPlex® and Cell ID™ Systems and kits from Life Technologies to facilitate a smoother and faster adoption of Promega STR products. The following information highlights the primary differences in the handling and use of Life Technologies AmpFlSTR® kits and Promega PowerPlex® 16 HS, 18D, 21 and Cell ID™ Systems. We recommend that you consider these points carefully when implementing Promega systems to obtain optimal data quality.

11113LATable 1. Comparison of STR Loci Amplified by Promega and Life Technologies STR Kits.

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Promega Matrix Standards and Spectral Calibration

Unlike matrix standards from Life Technologies, which are supplied premixed, Promega 4-dye and 5-dye matrix standards are supplied in separate tubes, one for each color in the dye set. These standards must be diluted in water (supplied with the kit) prior to mixing with Hi-Di™ formamide. Table 2 provides the catalog numbers and technical manual numbers for the matrix standards required for each instrument and kit type.

Table 2. Catalog Numbers and Technical Manual Numbers for Matrix Standards by Instrument and Kit Type.
Matrix Standards Catalog Number Technical Bulletin STR System Instrument Compatibility
PowerPlex® Matrix Standards, 310 DG4640 PowerPlex® Matrix Standards, 310, Technical Bulletin TBD021 PowerPlex® 16 HS System and Cell ID™ System ABI PRISM® 310 Genetic Analyzer
PowerPlex® Matrix Standards, 3100/31301 DG4650 PowerPlex® Matrix Standards, 3100/3130, Technical Bulletin TBD022 PowerPlex® 16 HS System and Cell ID™ System ABI PRISM® 3100 and 3100-Avant and Applied Biosystems® 3130, 3130xl, 3500 and 3500xL Genetic Analyzers
PowerPlex® 5-Dye Matrix Standards, 310 DG4600 PowerPlex® 5-Dye Matrix Standards, 310, Technical Bulletin TBD023 PowerPlex® 18D and 21 Systems ABI PRISM® 310 Genetic Analyzer
PowerPlex® 5-Dye Matrix Standards, 3100/31302 DG4700 PowerPlex® 5-Dye Matrix Standards, 3100/3130, Technical Bulletin TBD024 PowerPlex® 18D and 21 Systems ABI PRISM® 3100 and 3100-Avant and Applied Biosystems® 3130, 3130xl, 3500 and 3500xL Genetic Analyzers
1These matrix standards can be used to perform a spectral calibration for the 3730 series of instruments for Promega 4-dye chemistries using the Any4Dye calibration option.
2These matrix standards can be used to perform a spectral calibration for the 3730 series of instruments for Promega 5-dye chemistries using the G5-RCT option.

 

PowerPlex® System Handling and Use

Storage Conditions: Promega PowerPlex® 16 HS, 18D and 21 Systems and Cell ID™ System are shipped on dry ice and stored at –30°C to –10°C in a nonfrost-free freezer, whereas Life Technologies STR kits arrive frozen and are stored at 4°C after the first thaw. For these PowerPlex® Systems, it is possible to store pre-amplification reagents (master mix and primer pair mix) at 2–10°C for up to 1 week without loss of activity. For long-term storage, however, we recommend that reagents be placed at –20°C.

Mixing: The master mixes and primer pair mixes included in PowerPlex® and Cell ID™ Systems require thorough vortexing after thawing, as described in the technical manuals. Vortexing the reagents for the full length of time recommended in the protocol is important. Avoid centrifuging the vortexed reagents. Manually shake tubes down to collect the master mix, primer pair mix and PCR amplification mix at the bottom of the tubes. Inadequate vortexing or centrifuging after vortexing can result in suboptimal data quality.

Input DNA Amount: Promega PowerPlex® and Cell ID™ Systems may require different input amounts of DNA than Life Technologies STR kits. See Table 3 and the appropriate PowerPlex® Technical Manual for guidelines on recommended amounts of DNA template.

Table 3. Recommended Amount of DNA Template for the PowerPlex® and Cell ID™ Systems.
STR System Recommended Amount of DNA Template Technical Manual
PowerPlex® 16 HS System 0.5–1.0ng PowerPlex® 16 HS System Technical Manual TMD022
PowerPlex® 18D System1 1.0ng PowerPlex® 18D System Technical Manual TMD031
PowerPlex® 21 System 0.5ng PowerPlex® 21 System Technical Manual TMD034
Cell ID™ System 2ng Cell IDSystem Technical Manual TM074
1The PowerPlex® 18D System was optimized for direct amplification of common forensic database samples such as blood spotted onto unwashed FTA® cards. The use of extracted DNA with this kit may result in unbalanced profiles.

Positive Control DNA: Until recently, the PowerPlex® 16 HS System included the same positive control DNA as that in many Life Technologies STR kits (i.e., 9947A DNA). However, Promega scientists developed a new positive control DNA, the 2800M Control DNA, which is derived from human blood rather than a cell line, and this control DNA is now provided in the PowerPlex® 16 HS, 18D and 21 Systems. The 2800M Control DNA is supplied at a concentration of 10ng/µl and is often diluted prior to use.

The Cell ID™ System includes K562 DNA as the positive control DNA.

Thermal Cycling Conditions: Promega PowerPlex® and Cell ID™ Systems employ different thermal cycling parameters than those required by Life Technologies STR kits. Ensure that the recommended thermal cycling parameters are programmed for the specific kit, sample type and sample substrate as described in the appropriate technical manual (see Table 3). Note that for some systems such as the PowerPlex® 16 HS and Cell ID™ Systems, optimal performance requires the ability to slow the ramp rates down from the ramp speed dictated by the 9600 emulation mode. This ability to slow the ramp rate is critical for optimal balance of the kit. See the Thermal Cycling section of the appropriate technical manual for specific details. It is also important to note that when using the GeneAmp® PCR System 9700 thermal cycler with an aluminum block you may not be able to select max mode, which is the required ramp speed for some Promega systems such as the PowerPlex® 21 System.

Size Standards: An internal lane standard is included in Promega PowerPlex® and Cell ID™ Systems. The corresponding size standard is not included in Life Technologies STR kits, so the additional cost of the Life Technologies size standard should be considered when making side-by-side comparisons.

Monoplex Primers: For PowerPlex® 16 HS loci, monoplex primers are available for individual purchase. LifeTechnologies does not offer monoplex primers.

General Considerations

PowerPlex® 16 HS System

  • A 4-dye kit that uses the Internal Lane Standard 600 (ILS 600)
  • Amplification of 15 STR loci and Amelogenin. The D2S1338 and D19S433 loci are not present in this kit, but Promega has the capability to manufacture primer pairs for these loci as custom monoplexes on request
  • Hot-start DNA polymerase and optimized master mix included with the kit
  • Protocols for the GeneAmp® PCR System 9700 thermal cycler with a gold or silver heat block or a thermal cycler with the ability to program the ramp rate as directed in the PowerPlex® 16 HS System Technical Manual TMD022
  • Direct-amplification compatibility through the use of additional solutions (AmpSolution™ Reagent and either the SwabSolution™ Reagent for swabs or PunchSolution™ Reagent for nonFTA storage card punches)
  • No artifacts detected in the bin regions of any loci in initial tests of the PowerPlex® 16 HS System using POP-6™ and POP-7™ polymers. When using POP-7™ polymer, one artifact appears at the D3S1358 locus but is not in a bin location for any instrument or array length. See Figure 1.
    11120TA_870pxFigure 1. The PowerPlex® 16 HS System artifact when using POP-7™ polymer. The D3S1358 allelic ladder from the PowerPlex® 16 HS System was analyzed using an Applied Biosystems® 3130xl Genetic Analyzer, 36cm array and POP-4™ (Panel A), POP-6™ (Panel B) or POP-7™ (Panel C) polymer. Note that with POP-7™ polymer, the artifact runs within the locus but not in a bin location.

    The D3S1358 allelic ladder from the PowerPlex® 16 HS System was analyzed using an Applied Biosystems® 3130xl Genetic Analyzer, 36cm array and POP-4™ (Panel A), POP-6™ (Panel B) or POP-7™ (Panel C) polymer. Note that with POP-7™ polymer, the artifact runs within the locus but not in a bin location.

    /~/media/images/resources/figures/11100-11199/11120ta_870px.jpg?la=en

PowerPlex® 21 System

  • A 5-dye kit that uses the CC5 Internal Lane Standard 500 (CC5 ILS 500)
  • Amplification of 20 STR loci and Amelogenin
  • Hot-start DNA polymerase and optimized master mix included with the kit
  • Protocols for the GeneAmp® PCR System 9700 thermal cycler with gold or silver heat block programmed with Max mode as the ramp rate
  • Compatibility with both amplification of extracted DNA and direct amplification of DNA on FTA® storage card punches, SwabSolution™-treated buccal swabs and PunchSolution™-treated nonFTA storage card punches
  • Additional artifacts at D3S1358 and D16S539 when PowerPlex® 21-amplified products are analyzed with POP-7™ polymer. See Figure 2.
    11118TA_660pxFigure 2. Known artifacts with the PowerPlex® 21 System in D3S1358 and D16D539 with an Applied Biosystems® 3500 Genetic Analyzer, 50cm capillary and POP-7™ polymer.

    /~/media/images/resources/figures/11100-11199/11118ta_660px.jpg?la=en

PowerPlex® 18D System

  • A 5-dye kit that uses the CC5 Internal Lane Standard 500 (CC5 ILS 500)
  • Amplification of all loci in the PowerPlex® 16 HS System plus D2S1338 and D19S433
  • Hot-start DNA polymerase and optimized master mix included with the kit
  • Protocols for the GeneAmp® PCR System 9700 thermal cycler with gold or silver heat block run in 9600 emulation ramp rate mode
  • Protocols for both direct amplification and amplification of extracted DNA. However, amplification of extracted DNA can produce unbalanced results, so the PowerPlex® 18D System is not recommended for extracted DNA samples that require sensitive mixture detection
  • An artifact occurs at the D3S1358 locus when PowerPlex® 18D-amplified products are analyzed with POP-7™ polymer. See Figure 3.
    11119TA_900pxFigure 3. A known artifact with the PowerPlex® 18D System in D3S1358 with the Applied Biosystems 3500 Genetic Analyzer, 50cm capillary and POP-7™ polymer.

    /~/media/images/resources/figures/11100-11199/11119ta_900px.jpg?la=en

Cell ID™ System

  • A 4-dye kit that uses the Internal Lane Standard 600 (ILS 600)
  • Amplification of all eight core loci needed for cell line authentication by historical ATCC standards, which specify amplification of D5S818, D13S317, D7S820, D16S539, vWA, TH01, TPOX and CSF1PO
  • Protocols for the GeneAmp® PCR System 9700 thermal cycler with gold or silver heat block or a thermal cycler with the ability to program ramp rate as directed in the Cell IDSystem Technical Manual TM074
  • No artifacts in initial tests with the Cell ID™ System using POP-6™ and POP-7™ polymers on the Applied Biosystems® 3130, 3500 and 3730 instruments

Other Considerations

  • In some cases when data are generated using POP-7™ polymer, sample alleles are called as “spikes” when analyzed using GeneMapper® ID-X software, version 1.3 and earlier.
  • To analyze data in GeneMapper® software, versions 4.0 and 4.1, additional analysis files are required. For more information, contact Promega Technical Services at: genetic@promega.com

Note: Artifacts that appear when using POP-7™ polymer appear in the marker regions as a result of the separation properties of the polymer. Similar artifacts have been reported by Life Technologies when running Identifiler® reactions on an Applied Biosystems® 3730 DNA Analyzer with a 36cm capillary and POP-7™ polymer (1) . In some cases, artifacts increase in height with longer storage of the amplicon at 4°C. The size of each artifact (in bases) does not change significantly among instrument types.

References

  1. Applied Biosystems 3730 DNA Analyzer Human Identification Validation Report, Applied Biosystems

PowerPlex is a registered trademark of Promega Corporation. AmpSolution, Cell ID, PunchSolution and SwabSolution are trademarks of Promega Corporation.

5-FAM, JOE, NED, Hi-Di, POP-4, POP-6 and POP-7 are trademarks of Applera Corporation. ABI PRISM, AmpFlSTR, Cofiler, Identifiler and Profiler are registered trademarks of Applera Corporation. Applied Biosystems, GeneMapper and PET are registered trademarks of Applied Biosystems. FTA is a registered trademark of Flinders Technologies, Pty, Ltd., and is licensed to Whatman. GeneAmp is a registered trademark of Roche Molecular Systems, Inc. VIC is a registered trademark of Perkin-Elmer Corporation.

Figures

11120TA_870pxFigure 1. The PowerPlex® 16 HS System artifact when using POP-7™ polymer. The D3S1358 allelic ladder from the PowerPlex® 16 HS System was analyzed using an Applied Biosystems® 3130xl Genetic Analyzer, 36cm array and POP-4™ (Panel A), POP-6™ (Panel B) or POP-7™ (Panel C) polymer. Note that with POP-7™ polymer, the artifact runs within the locus but not in a bin location.

The D3S1358 allelic ladder from the PowerPlex® 16 HS System was analyzed using an Applied Biosystems® 3130xl Genetic Analyzer, 36cm array and POP-4™ (Panel A), POP-6™ (Panel B) or POP-7™ (Panel C) polymer. Note that with POP-7™ polymer, the artifact runs within the locus but not in a bin location.

/~/media/images/resources/figures/11100-11199/11120ta_870px.jpg?la=en
11118TA_660pxFigure 2. Known artifacts with the PowerPlex® 21 System in D3S1358 and D16D539 with an Applied Biosystems® 3500 Genetic Analyzer, 50cm capillary and POP-7™ polymer.

/~/media/images/resources/figures/11100-11199/11118ta_660px.jpg?la=en
11119TA_900pxFigure 3. A known artifact with the PowerPlex® 18D System in D3S1358 with the Applied Biosystems 3500 Genetic Analyzer, 50cm capillary and POP-7™ polymer.

/~/media/images/resources/figures/11100-11199/11119ta_900px.jpg?la=en

Tables

11113LATable 1. Comparison of STR Loci Amplified by Promega and Life Technologies STR Kits.

/~/media/images/resources/tables/11100 11199/11113la.jpg?la=en

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