Changing needs and growing demands of forensic DNA laboratories that aim to reduce sample-processing times and meet certain international standards, such as those recommended by EDNAP, stimulate both laboratories and manufacturers to seek new ways and processes to quickly achieve robust and reliable results. Many strategic improvements of laboratory processes can be implemented to optimize work conditions, but technological change is always necessary to bring this process to a higher level. With this vision, the Forensic Genetic Unit of the Legal Medical Service of Valparaiso evaluated the new PowerPlex® 18D System, leading to significant operational improvements in processing DNA samples. We believe that implementing the PowerPlex® 18D System will provide the necessary technological changes in our laboratory to make a breakthrough in the efficiency of our routine analysis. This new system allows direct amplification of short tandem repeats (STRs) using either two punches from a buccal sample or one punch from blood on a Whatman FTA® card in less than 90 minutes. This is a 50% reduction in amplification time compared with current systems used in our laboratory and eliminates the need for extraction of genetic material. The PowerPlex® 18D System contains all of the loci in the original PowerPlex® 16 System plus two additional loci, D2S1338 and D19S433, which increase the power of discrimination of the current systems used for routine analysis in our laboratory
The PowerPlex® 18D System was developed using multicapillary platforms, including the ABI PRISM® 3100 and 3100-Avant and Applied Biosystems® 3130, 3130xl, 3500 and 3500xL Genetic Analyzers
. Since we are from a small regional laboratory that uses the ABI PRISM® 310 Genetic Analyzer, it was important for us to evaluate the kit’s performance on the older, single-capillary platform. While the ABI PRISM® 310 Genetic Analyzer requires a much longer analysis time than multicapillary genetic analyzers for the same number of samples, the direct-amplification capabilities and the associated shorter amplification times for the PowerPlex® 18D System reduce the total analysis time greatly and allow small laboratories to reach higher levels of efficiency without necessarily investing in costly changes to capillary electrophoresis platforms.
The laboratories of the Legal Medical Service of Chile routinely use the AmpFlSTR® Identifiler® kit (Applied Biosystems), with the PowerPlex® 16 System as a complementary system to increase the power of exclusion in complex paternity cases. Including the D2S1338 and D19S433 loci in the new PowerPlex® 18D System allows us to confirm any unusual genetic variants at these loci by comparing results obtained using the Identifiler® and PowerPlex® 18D Systems. Most importantly, use of the PowerPlex® 18D System by itself allows us to obtain in a single amplification the same power of exclusion that previously could only be achieved by combining AmpFlSTR® Identifiler® and PowerPlex® 16 Systems.
The goal of this work was to verify the usefulness of the PowerPlex® 18D System on our ABI PRISM® 310 platform and establish the best amplification conditions for different types of samples that arrive at our lab for routine analysis, including whole blood, blood on FTA® cards and buccal swabs on FTA® cards.
Materials and Methods
Sample Collection: Three samples were collected from each of five volunteers (A–E) from the Legal Medical Service of Valparaiso in the following formats:
- Whole blood sample collected in tubes obtained by venipuncture using EDTA as an anticoagulant. DNA was extracted using the automated Maxwell® 16 Instrument and DNA IQ™ Reference Sample Kit (Cat.# AS1040) following the manufacturer's instructions.
- Buccal swab sample using the Whatman Indicating FTA® Mini Card following the manufacturer's instructions.
- Blood sample obtained from the fingertip using Accu-Chek® lancet, then deposited onto an FTA® card Genecard according to the manufacturer’s instructions.
Amplification: Amplifications were performed following the manufacturer's recommendations using a PCR amplification mix with the following volumes:
||Volume per Reaction
|Water, Amplification Grade
|PowerPlex® D 5X Master Mix
|PowerPlex® 18D 5X Primer Pair Mix
|Total reaction volume
For blood samples in the FTA® format, one 1.2mm punch was added to 25µl of PCR amplification mix. For buccal swab samples, two 1.2mm punches were added to 25µl of PCR amplification mix. For DNA samples extracted using the Maxwell® 16 Instrument, the volume of water was replaced by an equal volume of sample (15µl).
Amplification was performed using a GeneAmp® PCR System 9700 thermal cycler (Applied Biosystems). The thermal cycling protocol was the same for all samples, changing only the number of cycles in each case, and each sample type was amplified for 26, 27, 28 and 29 cycles. The protocol was as follows:
96°C for 2 minutes, then:
94°C for 10 seconds
60°C for 1 minute
for 26, 27, 28 or 29 cycles
60°C for 20 minutes
The positive control reaction was performed by diluting the 2800M Control DNA to 5ng/µl then adding 1µl of diluted DNA to the reaction.
Detection of Amplified Fragments: A loading cocktail was prepared to load samples onto the genetic analyzer by combining the following volumes:
||Volume per Sample
|CC5 Internal Lane Standard 500
We added 21µl of loading cocktail and 2µl of amplified sample to each tube. For the allelic ladder, we added 1.5µl to 21µl of loading cocktail.
Fragment detection was performed using the ABI PRISM® 310 Genetic Analyzer with the following run conditions:
Capillary length: 47cm × 50µm
Module: GS STR POP4 (1ml) G5.md5
Injection time: 5 seconds
Run time: 30 minutes
We used the following parameters in the default run module:
Inj. kV: 15.0
Run kV: 15.0
Run °C: 60
Results were analyzed using GeneMapper® ID software, version 3.2 (Applied Biosystems), using the panels and bins files downloaded from the Promega web site at: www.promega.com/geneticidtools/panels_bins/ and following the manufacturer's recommendations to generate an analysis method for this system. The matrix used for sample analysis was the PowerPlex® 5-Dye Matrix Standards, 310 (Cat.# DG4600), which was installed on the genetic analyzer as described in the PowerPlex® 5-Dye Matrix Standards, 310 Technical Bulletin TBD023.
Amplification was successful for all samples; however, the results differed depending on the sample type and number of cycles used. Amplification was considered successful when we obtained a complete genetic profile with mainly the following characteristics: signals greater than 200 relative fluorescence units (RFU), an intralocus peak height ratio at each locus greater than 70% and no extra peaks that can be considered as true alleles (i.e., no artifacts).
For blood samples on FTA® cards, successful genetic profiles were obtained using 26 and 27 cycles. The best results were obtained using 26 cycles (Figure 1). At 27 cycles, genetic profiles were acceptable, but in some cases peak heights were off scale. For all cases, peaks obtained with 28 and 29 cycles were off scale.
For buccal swab samples, successful genetic profiles were obtained using 27, 28 and 29 cycles, but 27 cycles provided the best results (Figure 2). At 26 cycles, some samples showed no or poor amplification.
For blood samples, successful profiles were obtained using 28 and 29 cycles. The best results were obtained using 29 cycles (Figure 3), with signal above 300RFU in all cases. However, we obtained acceptable genetic profiles with 28 cycles, but some loci had signals below 200RFU. In most cases, signals at 26 and 27 cycles were below 200RFU for several of the loci analyzed, and we observed an absence of alleles for some of them.
The genetic variants present for each individual remained constant regardless of cycle number, indicating that these peaks corresponded to true genetic variants present in each sample and were not artifacts caused by different amplification conditions. The positive control reaction generated the same genetic profile independently of the cycles used (Figure 4). Negative and extraction controls showed no signals at any of the cycle numbers (data not shown).
For most amplified samples, intralocus balance was greater than 70% for each locus amplified (Figures 5, 6 and 7). The average height of most peaks obtained at each amplified locus was greater than 200RFU. However, we observed signals below this value or even no signals for the same locus at different cycle numbers.
Figure 5. Intralocus balance percentage of heterozygote loci for blood samples A, B, C, D and E on FTA® cards at different cycles of amplification.
Note that at 26 and 27 cycles all loci exceed the threshold value of 70%.
In accordance with the objectives of this work, we can say that the PowerPlex® 18D System is a powerful and efficient amplification system. Analyses of different sample types were successful, and the genetic profiles obtained were concordant at each cycle number tested, confirming that the amplification conditions tested generated no artifacts that may affect the analysis of genetic variants present. To confirm the variants, we performed a small concordance analysis using blood samples extracted using the Maxwell® 16 Instrument and amplified using the AmpFlSTR® Identifiler® kit. The same genetic profiles were obtained with both systems.
The alleles obtained for each profile were easily identified, confirming that the run conditions selected for the ABI PRISM® 310 Genetic Analyzer were appropriate, the run times were adequate to resolve all peaks and the panels and bins recommended by the manufacturer worked properly. All alleles in the allelic ladder and tested samples fell in the correct corresponding bin. The panels and bins didn’t create any software conflicts with other analysis methods for other kits.
The run conditions used for the genetic analyzer worked well; we obtained clear signals with no truncated or failed runs. However, this will depend on the sensitivity of the genetic analyzer used. The selected matrix was implemented with no problems. We must mention that the PowerPlex® 18D System was not designed for the ABI PRISM® 310 Genetic Analyzer, so there were no manufacturer's recommendations for this instrument. We chose to model the current laboratory run conditions for the PowerPlex® 16 System, combining them with the run conditions recommended by the manufacturer for the ABI PRISM® 3100 and 3100-Avant Genetic Analyzers and the PowerPlex® 18D module used to run AmpFlSTR® Identifiler® kit, which was optimal for data collection.
For blood samples on FTA® cards, we obtained signals for all amplified loci for all cycle numbers tested, and intralocus balance was greater than 70% for the majority of cases (Figure 5). We found that 26 cycles gave the best amplification results because the samples had better interlocus balance compared to results obtained with other conditions after discarding the others that had off-scale peaks. Note that at 27 cycles optimal results were also obtained, with just a few of the samples showing signals that were out of range.
For buccal swab samples, optimal signals were obtained for all cycle numbers tested except one sample. The observed intralocus balance was greater than 70% for the majority of cases (Figure 6). We obtained the best results with 27, 28 and 29 cycles but chose 27 cycles because the genetic profiles obtained were complete and optimal for all samples analyzed. We recommend the use of 27 cycles for both blood samples on FTA® cards (most commonly used) and buccal swabs, making it possible to amplify both sample types at the same time in situations in which laboratory work makes it necessary.
For DNA samples extracted using the Maxwell® 16 Instrument, successful profiles were obtained only with 28 and 29 cycles of amplification but not for 26 and 27 cycles, which resulted in no signal or significantly low signal. Also, the best intralocus balance was obtained with 28 and 29 cycles of amplification (Figure 7). We defined the best amplification conditions as 29 cycles, which ensured successful amplification for all loci analyzed.
The Forensic Medical Service of Valparaiso, specifically the Forensic Genetics Unit, thanks Promega Corporation, with its representative in Chile Fermelo SA, for the invitation to evaluate the PowerPlex® 18D System.
We also thank the following:
The head of the Legal Medical Service of Valparaiso, Doctor Gabriel Zamora S, and the head of Laboratories, Viviana Cisternas A, for their support for the development of this initiative.
The Carlos Ybar Institute, represented by its Technical Secretary Doctor Gianna Gatti, for supporting this research and granting permission to do so.
The head of Forensic Genetics Unit of Santiago, Mr. Fabian Moreno Ch, for supporting this work and hoping that this could be done at the Forensic Genetics Unit of Valparaiso.
And especially Paulina Vega Celedón, Biochemistry student at the Pontifical Catholic University of Valparaiso, for his selfless work and tremendous efforts to carry out this work. Thanks Pauli!