|Year : 2015 | Volume
| Issue : 1 | Page : 8-15
Validation of the DNATyper™15 PCR Genotyping System for Forensic Application
Jian Ye1, Chengtao Jiang2, Xingchun Zhao1, Le Wang1, Caixia Li1, Anquan Ji3, Li Yuan4, Jing Sun1, Shuaifeng Chen5
1 Institute of Forensic Science, Ministry of Public Security; Beijing Engineering Research Center of Crime Scene Evidence Examination; Key Laboratory of Forensic Genetics, Ministry of Public Security, Beijing, China
2 Fada Institute of Forensic Medicine and Science, Beijing, China
3 Institute of Forensic Science, Ministry of Public Security, Beijing, China
4 Fada Institute of Forensic Medicine and Science; Key Laboratory of Evidence Science, China University of Political Science and Law, Ministry of Education, Beijing, China
5 Department of Investigation, People's Public Security University of China, Beijing, China
|Date of Web Publication||29-May-2015|
Beijing No. 3817 Letterbox, Beijing - 100038
Source of Support: None, Conflict of Interest: None
We describe the optimization and validation of the DNATyper™15 multiplex polymerase chain reaction (PCR) genotyping system for autosomal short tandem repeat (STR) amplification at 14 autosomal loci (D6S1043, D21S11, D7S820, CSF1PO, D2S1338, D3S1358, D13S317, D8S1179, D16S539, Penta E, D5S818, vWA, D18S51, and FGA) and amelogenin, a sex-determining locus. Several DNATyper™15 assay variables were optimized, including hot start Taq polymerase concentration, Taq polymerase activation time, magnesium concentration, primer concentration, annealing temperature, reaction volume, and cycle number. The performance of the assay was validated with respect to species specificity, sensitivity to template concentration, stability, accuracy, influence of the DNA extraction methods, and the ability to genotype the mixture samples. The performance of the DNATyper™15 system on casework samples was compared with that of two widely used STR amplification kits, Identifiler™ (Applied Biosystems, Carlsbad, CA, USA) and PowerPlex 16 ® (Promega, Madison, WI, USA). The conditions for PCR-based DNATyper™15 genotyping were optimized. Contamination from forensically relevant nonhuman DNA was not found to impact genotyping results, and full profiles were generated for all the reactions containing ≥ 0.125 ng of DNA template. No significant difference in performance was observed even after the DNATyper™15 assay components were subjected to 20 freeze-thaw cycles. The performances of DNATyper™15, Identifiler™, and PowerPlex 16 ® were comparable in terms of sensitivity and the ability to genotype the mixed samples and case-type samples, with the assays giving the same genotyping results for all the shared loci. The DNA extraction methods did not affect the performance of any of the systems. Our results demonstrate that the DNATyper™15 system is suitable for genotyping in both forensic DNA database work and case-type samples.
Keywords: DNATyper™15, kit, short tandem repeats (STRs), validation
|How to cite this article:|
Ye J, Jiang C, Zhao X, Wang L, Li C, Ji A, Yuan L, Sun J, Chen S. Validation of the DNATyper™15 PCR Genotyping System for Forensic Application. J Forensic Sci Med 2015;1:8-15
|How to cite this URL:|
Ye J, Jiang C, Zhao X, Wang L, Li C, Ji A, Yuan L, Sun J, Chen S. Validation of the DNATyper™15 PCR Genotyping System for Forensic Application. J Forensic Sci Med [serial online] 2015 [cited 2019 Aug 25];1:8-15. Available from: http://www.jfsmonline.com/text.asp?2015/1/1/8/157903
| Introduction|| |
Short tandem repeat (STR) genetic loci consist of short, repetitive sequence elements of 3-7 base pairs. Widely distributed throughout the human genome, these repeats are rich sources of polymorphisms, which can be analyzed using the polymerase chain reaction (PCR). 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. Multiplex STR analysis has emerged as the dominant forensic DNA-based identification method, because it can use subnanogram quantities of DNA to yield highly discriminatory, easily interpreted results in a couple of hours. ,,,,,,,,,
The DNATyper™ 15 system allows the coamplification and three-color detection of fifteen loci [amplification products of D6S1043, D7S820, D21S11, CSF1PO, and D2S1338 are labeled with fluorescein (FAM), while D5S818, vWA, D18S51, FGA, D3S1358, D13S317, D8S1179, D16S539, amelogenin, and Penta E amplicons are labeled with tetramethylrhodamine (TAMRA) and 4',5'-dichloro-2',7'- dimethoxy-fluorescein (JOE)]. All the 15 loci are simultaneously amplified in a single reaction tube and analyzed in a single gel lane. Labeling of genetic loci with different fluorescent dyes in the DNATyper™ 15 system is creatively arranged, improving upon the existing 13-loci pattern of the Combined DNA Index System (CODIS), which is recommended by the Federal Bureau of Investigation (FBI), and is closer to the genetic characteristics of the Chinese population. 
Before a new STR multiplex genotyping system can be routinely employed in human identity testing, extensive validation is required to ensure its reliability. In this study, we evaluated the species specificity, sensitivity, stability, accuracy, and optimal protocol of the DNATyper™ 15 system, and compared it with the commercially available alternatives. Our results demonstrate the robustness of this system, indicating the potential for using the DNATyper™15 kit (Institute of Forensic Science, Ministry of Public Security, Beijing, China) with casework samples in forensic laboratories.
| Materials and Methods|| |
Samples and extraction protocols
We have a long-term, cooperative relationship with 34 DNA analysis laboratories (see Supporting Information), which assisted in the collection of blood samples. Written informed consent was given by all the participants involved in the study. We obtained ethical approval for the study from the Ethical Review Board of the Institute of Forensic Science of China. 9947A control genomic DNA was purchased from Promega. Primate and nonprimate genomic DNA samples were obtained from the DNA laboratory at the Institute of Forensic Science (Beijing, People's Republic of China). Genomic DNA extraction was performed using either phenol-chloroform  or the Chelex-100  method.
DNA laboratories in the following departments in China assisted in collecting blood DNA samples for this study:
- Beijing Municipal Public Security Bureau
- Shanghai Municipal Public Security Bureau
- Department of public security of Zhejiang Province
- Department of public security of Guangdong Province
- Department of public security of Hubei Province
- Department of public security of Hunan Province
- Department of public security of Henan Province
- Department of public security of Hebei Province
- Department of public security of Shandong Province
- Zibo Municipal Public Security Bureau
- Taian Municipal Public Security Bureau
- Dezhou Municipal Public Security Bureau
- Anshan Municipal Public Security Bureau
- Department of public security of Jiangsu Province
- Nanjing Municipal Public Security Bureau
- Zhenjiang Municipal Public Security Bureau
- Taizhou Municipal Public Security Bureau
- Nantong Municipal Public Security Bureau
- Tianjin Municipal Public Security Bureau
- Department of public security of Liaoning Province
- Hangzhou Municipal Public Security Bureau
- Guangzhou Municipal Public Security Bureau
- Yichang Municipal Public Security Bureau
- Department of public security of Jiangxi Province
- Zhengzhou Municipal Public Security Bureau
- Wuhan Municipal Public Security Bureau
- Weifang Municipal Public Security Bureau
- Jinan Municipal Public Security Bureau
- Jining Municipal Public Security Bureau
- Institute of Forensic Science, Ministry of Public Security
- Suzhou Municipal Public Security Bureau
- Changzhou Municipal Public Security Bureau
- Yangzhou Municipal Public Security Bureau
- Yancheng Municipal Public Security Bureau
Unless otherwise stated, a PCR master mix was prepared for all amplifications, including each reaction, 4 μL of DNATyper™ 15 PCR reaction mix, 0.4 μL of Taq DNA polymerase, 2 μL of DNATyper™ 15 primer, and 11.6 μL of double distilled water (ddH 2 O). Then 2 μL of genomic DNA was added to 18 μL of the master mix, so that the final template input was in the range 0.5-1.25 ng, with a total reaction volume of 20 μL. PCR reactions were performed in either a GeneAmp®PCR System 9600 (Applied Biosystems) or a GeneAmp®PCR System 9700 operating in 9600 emulation mode. The thermal profile consisted of a 5-min incubation at 95°C, followed by 28 or 30 cycles of three-step PCR with 1 min each at 94°C, 59°C, and 70°C, and a 60-min hold at 60°C. A final hold at 25°C was added if the PCR product was to remain in the thermal cycler for ≤ 18 h, and, if > 18 h, a 4°C hold was used.
Electrophoresis, detection, and analysis
Analysis of amplification products was performed primarily on ABI PRISM ® 310, 3100, and 3130XL Genetic Analyzers (Applied Biosystems, Foster City, USA), using 3-s or 5-s injection times. Electrophoresis used performance-optimized polymer 4 POP4 and 47-cm capillaries from Applied Biosystems. Generally, 1 μL of amplified sample containing carboxy-X-rhodamine (CXR) 488 was added to 24-μL-deionized formamide. The samples were denatured for 3 min at 95°C followed by cooling on ice, prior to electrophoresis. The DNATyper™ 15 kit employs four fluorescent dyes. Spectral resolution was established by DNATyper™ 1 5 matrix fluorescence (FL)-hexachloro-fluorescein (HEX)- carboxytetramethylrhodamine (TMR)-ROX, enabling the evaluation of each fluorescent dye. All the analyses utilized the DNATyper™ 500 size standard and the allelic ladder mix [Figure 1] provided with the DNATyper™ 15 kit. Initial fragment sizing was performed using the GeneScan ® software (Applied Biosystems). Allele calling was performed by a DNATyper™ 15 Macro operating within the Genotyper ® software or GeneMapper ® ID v3.2 software program, (Applied Biosystems).
|Figure 1: Allelic ladder mix of the DNATyperTM15 kit. Panel A: The blue fluorescein-labeled allelic ladder components (D6S1043, D7S820, D21S11, CSF1PO, and D2S1338) and their allele designations. Panel B: The green JOE-labeled allelic ladder components (D3S1358, D13S317, D8S1179, D16S539, and Penta E) and their allele designations. Panel C: The yellow TMR-labeled allelic ladder components (D5S818, vWA, D18S51, FGA, and amelogenin) and their allele designations.|
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Species specificity and sensitivity
The DNATyper™15 primer kits are designed to be primate-specific, with no cross-reaction with other animals or microbial species. PCR reactions were performed with 1 ng of genomic DNA from macaque and gibbon to determine the amplification profiles for these primate species. Reactions for nonprimate species (sheep, donkey, dog, horse, and cow) were performed with 2.5-10 ng of genomic DNA.
Assessing the amplification performance with a range of DNA input amounts is helpful in understanding the potential interpretational limitations of a PCR-based genotyping system. Triplicate amplifications were performed using a dilution series of human 9947A genomic DNA template samples, giving 2.0 ng, 1.0 ng, 0.5 ng, 0.25 ng, 0.125 ng, and 0.0625 ng template per reaction.
Population samples for STR polymorphism analysis
Blood samples were collected from a total of 290 unrelated healthy individuals from the Han Chinese population, the largest in China. All samples of the volunteers were genotyped with Identifiler™ . Genomic DNA was extracted using the Chelex-100 protocol. The quantity of recovered DNA was determined by the spectrophotometric method.
| Results and Discussion|| |
PCR-based procedures: Taq polymerase titration
DNA polymerase has a vital role in PCR-based STR genotyping strategies, influencing the amplicon yield and locus-to-locus signal balance. We evaluated the optimal concentration of Taq polymerase. Incremental amounts (0.5-8 U) of Taq polymerase were assessed in PCR with 28 cycles. At the lowest and highest levels tested, peak heights were lower and higher, respectively, than those at the standard input level, without affecting the completeness of the profiles [Figure 2]. Allele dropout was not observed under any of the conditions tested. Similar results were observed when testing AmpliTaq Gold DNA Polymerase ( Life Technologies, Applied Biosystems, Foster City, USA) concentrations in the range of ± 10% around the recommended 2 U in 20 μL reaction volumes (data not shown).
|Figure 2: Variations of hot start Taq polymerase amount in the DNATyperTM15 kit. Representative electropherograms from amplification of 1 ng 9947A human genomic DNA. The panels depict 0.5 U, 1 U, 2 U, 4 U, and 8 U hot start Taq polymerase from top to bottom. The X-axis indicates base pair size, while the Y-axis indicates RFU.|
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PCR-based procedures: Taq polymerase activation time
Activation time of 3 min, 5 min, 8 min, 11 min, and 14 min were tested using 9947A female human control DNA and the DNATyper™ 15 standard PCR reaction mix (1 ng DNA in 20 μL of reaction mixture). Taq DNA polymerase was found to perform optimally with a 5-min activation time [Supplementary Figure 1].
PCR-based procedures: Titration of magnesium
As a cofactor of DNA polymerase, magnesium functions as a critical reagent for PCR amplifications and has important roles in both polymerase activity and specificity.  To examine the effect of magnesium variations, concentrations between 1 mM and 2 mM (1.25 mM recommended) were examined using 28 cycles and 1 ng of template DNA [Supplementary Figure 2]. An optimal balance between loci was seen at 1.25 mM magnesium. When the magnesium concentration was < 1.25 mM, some loci were amplified inefficiently. The dropout of one locus was seen in the reactions with 1 mM magnesium. Magnesium concentrations > 1.25 mM resulted in poor amplification balance and specificity, and produced a slight increase in the yield of smaller loci.
PCR-based procedures: Primer pair titration
The concentrations of the 32 primers in DNATyper™ 15 have been determined to produce equivalent signals for each locus. Although further adjustment of the primer balance is not necessary, overall primer input can slightly vary due to pipetting errors. Primer concentrations between 0.5X and 2X were tested with 30 cycles of PCR and 1 ng of template DNA to determine the effects of varying the total amount of primer on peak height and amplification balance between the loci [Supplementary Figure 3]. When the primer concentration was increased above 1X, smaller loci (in bases) were preferentially amplified. As the primer concentration was decreased below 1X, peak heights decreased for shorter amplicons and increased for longer amplicons. These data suggest that normal variability in pipetting precision will have a minimal effect but intentional changes in primer concentration may substantially influence the results.
PCR-based procedures: Annealing temperature
Changes to the annealing temperature of the amplification reaction can affect the specificity and balance of the amplified loci. To examine these effects, PCR was performed using annealing temperatures ranging from 55°C to the recommended 65°C, with 28 cycles and 1 ng of 9947A female human control template DNA [Figure 3]. Lower annealing temperatures did not cause locus dropout, but resulted in an increase in the yield of smaller amplicons.
|Figure 3: Variations of annealing temperature. Representative electropherograms from amplification of 1 ng 9947A human genomic DNA. The panels depict annealing temperature of 55°C, 57°C, 59°C, 61°C, and 63°C in order from top to bottom.|
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PCR-based procedures: Variation of reaction volume
Reduction of reaction volume, which is often used as a cost-saving measure, can alter the concentration of PCR reaction components. Varied reaction volumes were evaluated to determine their effects on amplification with DNATyper™ 15 [Supplementary Figure 4]. Two approaches can be followed in changing reaction volumes: keeping the template concentration constant or keeping the quantity of template constant. In this study, we chose to keep the concentration constant to reduce the effects of high template concentrations such as high background and saturation of the charge-coupled device (CCD) pixels that may lead to overwhelmed or incomplete spectral subtraction, resulting in phenomena known as pulldown or pullup. Reaction volumes ranged 10-40 μL (20 μL is the standard volume), with template titration at 1 ng/20 μL. As expected, signal intensity remained similar at all the tested volumes. Decreasing the reaction volume may be a useful adaptation for very low template protocols, as it effectively increases the concentration for a given amount of template. However, stochastic effects and corresponding increases in the concentration of inhibitors with low reaction volumes are potential concerns.
PCR-based procedures: Cycle number
The amplification cycle number provides flexibility to STR genotyping systems. Increasing the cycle number can increase sensitivity, while decreasing the cycle number may attenuate or balance signals when the DNA template is in excess. In order to optimize this parameter, we tested 27, 28, 29, 30, and 31 cycles for amplification of 1 ng of 9947A DNA. As expected, most peak heights increased with each additional PCR cycle. The results appeared very similar with 28, 29, and 30 cycles in terms of balance, although incrementally increased peak heights were observed with each added cycle [Supplementary Figure 5]. The highest cycle number (31 cycles) produced only a slight increase in the yield for the largest amplicon, whereas the smaller loci displayed a substantial increase in signal, leading to a notable imbalance between the loci.
Primers included in the DNATyper™ 15 kit are intended to genotype human DNA. Species-specific genetic differences and sound primer design help to ensure minimal cross-reactivity with nonhuman sources of DNA, many of which can be found in forensic laboratories. In this study, we assessed the potential to detect DNA from eight nonhuman species (gibbon, sheep, donkey, dog, macaque, cow, horse, and mouse). Amplification was observed only among the primates, with one notable exception. 1 ng of primate DNA (macaque and gibbon) produced partial profiles on the odonthyalus protein gene (amelogenin). We saw no detectable DNA profiles in the nonprimates (sheep, donkey, dog, horse, and cow). The remainder of the tested species exhibited no amplification. These results show that contamination from forensically relevant nonhuman DNA should not affect genotyping with DNATyper™ 15. Representative electropherograms for several species of template DNA are illustrated in Supplementary Figure 6].
We attempted to determine the quantity of DNA template required to produce reliable genotyping results using the DNATyper™ 15 kit. The results indicated that the peak height decreases progressively with decreasing amounts of template DNA. For triplicate amplifications performed on control DNA 9947A, with input ranging 0.0625-1 ng, all the data were onscale [Table 1]. Full profiles were generated for all but the lowest template level (0.0625 ng), assuming a 50 relative fluorescence units (RFU)-peak amplitude threshold. All the 14 STR loci were amplified with 0.125 ng template DNA. The results were similar with Identifiler™ and PowerPlex 16 ® kits , as shown in [Figure 4].
|Figure 4: Sensitivity test. Representative electropherograms from amplification of 1 ng, 0.5 ng, 0.25 ng, 0.125 ng, and 0.0625 ng of 9947A human genomic DNA orderly displayed from top to bottom.|
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As part of the development of the DNATyper™ 15 system, freeze-thaw testing was conducted to confirm the robustness of amplification. No noticeable difference in genotyping was observed after the DNATyper™ 15 kit underwent 5, 10, 15, and 20 freeze-thaw cycles. DNATyper™ 15 kit performance after 20 freeze-thaw cycles is shown in Supplementary Figure 7].
Mixture analysis has an important role in many casework studies. To analyze the ability of the DNATyper™ 15 kit to provide reliable results from mixed-source samples, we prepared and tested a mixture series of 9947A and 9948 female and male human genomic DNA (Promega), respectively. This included 9947A: 9948 DNA ratios from 10:1 to 1:10, with the total DNA amount kept constant at 1 ng per reaction. As shown in [Figure 5], the increasing or decreasing trend of 9947A and 9948 DNA in genotype mixtures agrees well with the ratios in these mixture samples. As the ratios become more extreme, the percentage of minor alleles detected decreases, averaging about 50% detection at 10:1 and 1:10 ratios and about 17% at 19:1 and 1:19 ratios. However, there was a great deal of variation from one laboratory to another, depending on the sensitivity of the ABI PRISM ® 310 Genetic Analyzers, the amount of DNA used, and the RFU cutoff limit.
|Figure 5: Mixture studies with the DNATyperTM15 kit. Representative electropherograms from amplification of 1 ng of mixed 9947A female and 9948 male human genomic DNA at indicated ratios were shown.|
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Comparison with commercial kits
To compare the performance of the DNATyper™ 15 kit with other commercially available kits, we carried out STR genotyping experiments side-by-side with DNATyper™ 15, Identifiler™ , and PowerPlex 16 ® kits. Also, the casework samples were examined to determine if reliable results could be obtained with samples that are commonly encountered in forensic DNA testing. The DNATyper™ 15 kit successfully generated complete STR genotyping results from a cigarette butt, a semen stain, and costal cartilage [Supplementary Figure 8]. Our results show that DNATyper™ 15 performs equally well compared with commercially available kits, and all the three kits give the same genotyping results on the shared genetic loci. Different DNA extraction methods (Chelex-100, magnetic beads, or silica beads) did not result in observable effects on performance with any of the three kits (data not shown).
Accuracy of the DNATyper™ 15 Kit
Due to the influences of electrophoretic conditions such as ion intensity and equipment, slight differences can exist in DNA fragment and allelic ladder separation. To eliminate this kind of electrophoretic deviation, the general method is to set a tolerance sector (confidence interval) of 0.5 basic group at the gene stereotypic stage. Only if deviations remain within the 0.5 basic group scope, can the corresponding alleles be accurately genotyped. As [Figure 6] illustrates, through calculation of the differences between allele fragments and the allelic ladder, the deviations of all 2,399 alleles from 89 samples fell within the ± 0.5 basic group range, indicating that the DNATyper™ 15 kit has the capability of producing accurate genotyping.
|Figure 6: Precision of ILS 500. The size deviations of 89 samples were analyzed on the ABI PRISM 3100 Genetic Analyzer, (Applied Biosystems, Foster City, USA) and then the average fragment size (bases) of each allele was plotted against the tolerance sector.|
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Allele frequencies and forensic parameters for 14 STRs
The polymorphism analysis of the 14 autosomal STRs was studied in the Chinese Han population of China. All the studied STRs followed Hardy-Weinberg equilibrium. In Supplementary Table 1, we present allele frequencies derived from the population and the forensic efficiency values for each STR. The most informative loci in our dataset were D6S1043 and Penta E, with discrimination power values of 0.966 and 0.984, respectively. These two loci are not included in the 13 CODIS STR gene loci recommended by the FBI. Adoption of these highly informative loci, but not TH01 and TPOX that have relatively less power of discrimination, makes the DNATyper™ 15 kit better suited to the genetic characteristics of the Han Chinese population.
| Conclusion|| |
We have provided evidence that the DNATyper™ 15 system is robust in handling moderate changes to the preferred amplification protocol and to various sample sources. This multiplex system demonstrated a high level of sensitivity in obtaining complete genotypes, and is primarily limited by stochastic effects inherent in PCR amplification. Our results show that the 14 STR loci analyzed with the DNATyper™ 15 system are highly polymorphic, and can be useful for human identification and kinship analysis. Compared with other kits, this multiplex system appears to be more suitable for the Chinese population. Furthermore, the DNATyper™ 15 multiplex system is robust and reliable when applied to samples commonly found in a casework environment.
| Acknowledgement|| |
Funding was provided by the National Science and Technology Supporting Program during the 10 th 5-year plan period (2001BA801B02). We thank all our collaborators, the 34 DNA laboratories that kindly assisted in collecting blood samples, for their generous help.
[Additional file 1]
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
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