Journal of Forensic Science and Medicine

: 2020  |  Volume : 6  |  Issue : 3  |  Page : 79--88

Developmental validation of the EX16+22Y system

Xin Wang1, Weizhong Chen1, Jian Zhang1, Yang Cui1, Jingjing Chen1, Yunjie Shi1, Gao Wang2, Fayuan Li2, Yong Liu2, Ruhua Zhou1,  
1 Department of DNA Laboratory, Institute of Criminal Science and Technology, Suzhou Municipal Public Security Bureau, Xiangcheng, Suzhou, China
2 Department of Research and Development, AGCU ScienTech Incorporation, Huishan, Wuxi, China

Correspondence Address:
Ruhua Zhou
3998 Renmin Road, Xiangcheng, Suzhou


The EX16+22Y system is a polymerase chain reaction (PCR)-based amplification kit that enables typing of 15 autosomal short tandem repeat (STR) loci (i.e., D3S1358, D13S317, D7S820, D16S539, TPOX, TH01, D2S1338, CSF1PO, D19S433, vWA, D18S51, D21S11, D8S1179, D5S818, and FGA) and 22 widely used Y chromosome STR (Y-STR) loci (DYS391, DYS527a/b, DYS635, DYS458, DYS456, DYS385a/b, DYS438, DYS448, DYS437, DYS19, DYS576, DYS533, DYS393, DYS389I/II, DYS439, DYS392, Y_GATA_H4, DYS390, and DYS481) which contains 20 core Y-STR recommended by the Ministry of Public Security and amelogenin. This multiplex system was designed for the simultaneous analysis of amelogenin-Y allele mutation, single-source searches, kinship (including familial searching), mixture profiles, international data sharing, and other forensic applications. In this study, the multiplex system was validated for sensitivity of detection, species specificity, DNA mixtures, stability, sizing precision, stutter, reproducibility, and PCR-based conditions according to the Scientific Working Group on DNA Analysis Methods developmental validation guidelines and Chinese criteria for the human fluorescent STR multiplex PCR reagent. The results show that the EX16+22Y system is a robust and reliable amplification kit which can be used for human identification testing.

How to cite this article:
Wang X, Chen W, Zhang J, Cui Y, Chen J, Shi Y, Wang G, Li F, Liu Y, Zhou R. Developmental validation of the EX16+22Y system.J Forensic Sci Med 2020;6:79-88

How to cite this URL:
Wang X, Chen W, Zhang J, Cui Y, Chen J, Shi Y, Wang G, Li F, Liu Y, Zhou R. Developmental validation of the EX16+22Y system. J Forensic Sci Med [serial online] 2020 [cited 2020 Dec 5 ];6:79-88
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Full Text


Autosomal short tandem repeats (A-STRs) and Y chromosome STRs (Y-STRs) have been widely applied in forensic DNA analyses for many years. The rapid growth of DNA databases requires more markers to be included in multiplex kits to meet the challenges of adventitious hits, missing person identification, international data sharing, etc.[1],[2] With the rapidly development of current STR and capillary electrophoresis (CE) technology, the number of markers in one kit can multiplex up to about 25~30 STR loci. In recent years, a novel multiplex approach with combination of A-STRs and several Y-STRs has been presented to provide a higher kinship index with sufficient discriminating power for single-source and mixture comparisons compared with a similar number of only A-STR loci.[3],[4]

At present, Fusion (Promega, USA) and GlobalFiler (Life Technologies, USA) were designed to incorporate autosomal and few Y-STR loci, which are useful in individual identification and paternity testing. However, the utility of several Y-STR loci in one multiplex system is insufficiently powerful for kinship analyses and determining potential contributors of mixtures. More Y-STR loci are very useful tool in investigations of sexual assault, paternity and genealogical tests, and evolutionary studies.[5],[6],[7] It is necessary to design a new multiplex system which combines a dozer Y-STRs with available autosomal STRs.

In this study, we developed and validated the EX16+22Y system. The multiplex is a 6-dye system that co-amplifies 15 A-STR loci, 22 Y-STR loci, and amelogenin. The spectral configuration of the EX16+22Y system is as follows: DYS391, D3S1358, D13S317, D7S820, D16S539, DYS527a/b, DYS635, and DYS458 labeled with 6-FAM; DYS456, TPOX, TH01, D2S1338, CSF1PO, DYS385a/b, DYS438, and DYS448 labeled with HEX; DYS437, D19S433, vWA, DYS19, D18S51, and DYS576 labeled with SUM; amelogenin, D8S1179, D5S818, D21S11, FGA, and DYS533 labeled with LYN; and DYS393, DYS389I/II, DYS439, DYS392, Y_GATA_H4, DYS390, and DYS481 labeled with PUR [Figure 1]. The information of each locus is shown in [Table 1].{Figure 1}{Table 1}

The EX16+22Y system was performed to evaluate sensitivity, species specificity, DNA mixtures, stability, precision, stutter, reproducibility, and polymerase chain reaction (PCR)-based conditions using control DNA 9948, control DNA 9947A, and 2800M (Promega, WI, USA) and 007 (Life Technologies) according to FBI QAS (2009/2011), Scientific Working Group on DNA Analysis Methods developmental validation guidelines, and Chinese criteria.[8]

 Materials and Methods

DNA samples

The control 9948 and the control 9947A were purchased from OriGene, and the 2800M DNA was purchased from Promega. Direct amplification reactions were performed using one 1.2 mm punch from blood FTA® Cards and buccal indicating FTA® Cards.

Ethic clearence

DNA samples were collected with informed consent from 840 donors of Suzhou Han population, and the study was approved by the Ethics Committee of Southern Medical University.

Polymerase chain reaction amplification

Primer concentrations in the EX16+22Y system ranged from 0.11 to 0.48 μM. Amplifications were performed in a GeneAmp® PCR System 9700 thermal cycler (Life Technologies, CA, USA) using a 25 μL reaction volume, which contained 10 μL reaction mix, 5.0 U C-Taq DNA polymerase (AGCU, Wuxi, China), 5 μL 5×EX16+22Y primer set, 0.52 ng of pure DNA. The PCR conditions were 95°C for 2 min, 15 cycles of 94°C for 30s, 60°C for 1 min, and 72°C for 1 min, 15 cycles of 90°C for 30s, 58°C for 1 min, and 65°C for 1 min 20 s and a final extension at 60°C for 20 min. The reaction master mix consisted of 2.5 mM magnesium ion, 0.25 mM dNTPs, 50 mM Tris-HCl (pH 8.3), 50 mM KCl, 10 mM betaine, 1.2 mM bovine serum albumin, 6% glycerol, and 0.01% NaN3.

Sample electrophoresis and data analysis

Amplified PCR products were separated and detected on the Applied Biosystems 3130xl and 3500xl Genetic Analyzers (24-capillary) (Thermo Fisher Scientific) using the specified J6 variable binning modules. Spectral calibration was performed using J6 Dye set with the 6-Dye Matrix Standards (containing six DNA fragments labeled FAM, HEX, SUM, LYN, PUR, and SIZ, respectively). Electrophoresis samples were prepared by adding 1 μl of the PCR product or allelic ladder to 12 μl of Formamide/AGCU Marker SIZ-600 solution (0.5 μL of AGCU Marker SIZ-600 size standard and 11.5 μL of deionized Hi-Di™ Formamide, Thermo Fisher Scientific) (AGCU ScienTech, China). Before electrophoresis, the samples were denatured at 95°C for 3 min then chilled on ice for 1 min. Samples were injected at 3kV for 10s and electrophoresed at 15 kV for 1800s in Polymer-4 (Thermo Fisher Scientific) with a run temperature of 60°C on the Applied Biosystems 3130xl Genetic Analyzers, while samples processed on the 3500 xL Genetic Analyzers were injected at 1.2 kV for 24 s and electrophoresed at 13 kV for 1400 s in POP-4™ polymer with a run temperature of 60°C. GeneMapper ID-X Software v1.3 (Thermo Fisher Scientific, Foster City, CA, USA) was used to analyze results with 50 relative fluorescence units (RFU) of analytical threshold for 3130xl Genetic Analyzer and 175 RFU for 3500xL Genetic Analyzers.

Sensitivity studies

To evaluate the sensitivity of the EX16+22Y system, a serial dilution of DNA 2800M and 9948 from 31.25 pg to 1 ng was analyzed in triplicate. Full profile percentage was calculated for each 9948 and 2800M DNA amount.

Species specificity

The species specificity studies were conducted using 0.5 ng of input DNA from humans, chimpanzees, and macaques; 10 ng of input DNA from animals (cat, dog, pig, oxen, and chicken); and 10 ng of microbial DNA pool (Escherichia coli, Lactobacillus acidophilus, Streptococcus salivarius, Saccharomyces cerevisiae, and Enterococcus faecalis). These samples were kindly donated from Wuxi Zoo (Jiangsu, China). The quantities were determined using a Qubit® ssDNA Assay Kit with a Qubit® 3.0 Fluorometer (Invitrogen/Life Technologies, San Francisco, CA, USA).

DNA mixture study

Male/male mixtures were prepared using 9948 and 2800M human genomic DNA with mixture ratios of 1:1, 1:4, 1:9, and 1:19. Male/female mixtures were prepared using 9948 and 9947A (Promega, WI, USA) human genomic DNA with mixture ratios of 1:1, 1:4, 1:9, and 1:19. Each mixture was tested in triplicate. These mixed samples were formulated at a total concentration of 1 ng in 25 μL volume.

Stability studies

Several common forensic inhibitors were tested, including hematin, hemoglobin, humic acid, indigo, calcium ion, and ethylenediaminetetraacetic acid (EDTA). The quantity of 9948 DNA was constant at 0.5 ng, while the inhibitor concentrations varied: 20, 50, 80, 100, or 150 μmol/L of hematin; 50, 80,100, 150, or 200 μmol/L of hemoglobin; 20, 50, 80, 100, or 150 ng/μl of humic acid; 4, 5, 8, 10, or 12 mmol/L of indigo; 0.1, 0.2, 0.5, 0.7, or 1.0 mmol/L of calcium ions; and 0.1, 0.5, 0.8, 1.0, or 1.2 mmol/L of EDTA.

Sizing precision and stutter effects

The allele ladder was run on an Applied Biosystems 3500xL Genetic Analyzer. The average base-pair sizes and standard deviation (SD) were calculated for each allele in the allelic ladder (3500xL-24 injections) using the AGCU Marker SIZ-600 and GeneMapper ID-X v1.3 software.

Stutter peaks which are caused by slippage of the Taq polymerase during the elongation step are common artifacts observed during the DNA-STR amplification.[9],[10],[11] Peaks one repeat smaller or larger than the true allele were the most common stutter product. The proportion of the stutter peaks relative to the true allele was measured by dividing the stutter peaks by the true peak height. Five hundred and eighty-two samples were used for the stutter ratio calculation.


Reproducibility samples included buccal indicating FTA® Cards and blood FTA® Cards (GE Healthcare/Whatman) and extracted DNA from 24 male donors and 24 female donors. The DNA was extracted using magnetic beads (DNA IQTM System) on the Maxwell® 16 Research System (Promega). The quantity was determined using the Qubit® ssDNA Assay Kit. Four laboratories (i.e., Suzhou Public Security Bureau and two of its subinstitutes and Wuxi Public Security Bureau) were involved in the experiments.

Polymerase chain reaction condition study

The PCR condition studies included the test of cycle number, annealing temperature, final extension time, concentrations of reaction mix, Taq polymerase, and primer set.

 Results and Discussion

Sensitivity studies

The sensitivity of the EX16+22Y system was determined using a range of 9948 and 2800M DNA. We obtained full profiles from 0.125 ng DNA template as the analysis threshold was 175 RFU peak heights [Figure 2]. When both the 9948 and 2800M DNA decreased to 0.0625 ng, almost average 80% of the loci were called. For the amount of template DNA reduced to 0.03125ng, almost average 30% alleles observed were detected.{Figure 2}

Species specificity

Nonhuman genomic DNA samples were tested for cross-reaction with the EX16+22Y system. As seen with [Figure 3], some squatty peaks between 100 and 400 bp were observed in the profiles of primate DNA (chimpanzee and macaque) which were distinguishable from human genomic DNA profiles by their allele number of off-ladder. No peak was observed at 10 ng DNA per PCR for other common animals and the microbial pool.{Figure 3}

DNA mixture study

Forensic casework analysis usually involves mixture samples that contain the DNA from more than one individual. Therefore, it is very important to reliably interpret results from mixed-source samples. In this validation, male/male mixtures and male/female mixtures were tested and summarized, as listed in [Table 2]. All alleles were called for the 1:1 and 1:4 mixtures.{Table 2}

As the mixture ratio was increased (1:9 or 1:19), there was a decrease in the percentage of minor alleles that could be detected, because they were below the detection threshold (0.125 ng). For example, as male/male mixture ratio was 1:9, the alleles of the five loci including CSF1PO, DYS389I/II, DYS533, and DYS392 dropped out. The mixture studies show that this system can meet the requirement of the analysis for mix samples and Chinese criteria for the human fluorescent STR multiplex PCR reagent, which requires that a valid kit should be able to detect all the alleles for 1:4 mixtures.

Stability studies

Six common inhibitors (i.e., hematin, hemoglobin, humic acid, indigo, calcium ion, and EDTA) were tested with the EX16+22Y system. Full profiles were obtained with 20 and 50 μmol/L of hematin; 50 and 80 μmol/L of hemoglobin; 20 ng/μl of humic acid; 4, 5, 8, and 10 mmol/L of indigo; 0.1, 0.2, 0.5, and 0.7 mmol/L of calcium ion; and 0.5, 0.8, and 1.0 mmol/L of EDTA; when the concentration of hematin increased to 80 μmol/L, the DYS19, DYS392, DYS448, and DYS533 loci dropped out. When the concentration of hemoglobin increased to 100 μmol/L, the DYS19 and DYS392 loci dropped out. If the concentration further increased to 200 μmol/L, there were only 26.3% alleles called. When the concentration of humic acid increased to 50 ng/μl, the DYS19, DYS392, DYS533, Y_GATA_H4, and DYS390 loci dropped out. When the concentration of calcium ion increased to1.0 mmol/L and EDTA increased to 1.2 mmol/L, the DYS19, DYS392, and DYS533 loci dropped out [Table 3].{Table 3}

Sizing precision and stutter effects

Precision of the EX16+22Y system was evaluated on 3500 × L Genetic Analyzers. For each instrument, two full injections containing EX16+22Y system allelic ladder [Figure 4] were run and sized with the AGCU Marker SIZ-600. As expected, the largest allele sizes yield the greatest SD. For the 3500 × L Genetic Analyzers, the maximum SD of an allele was less than 0.2 bases [Figure 5]. These results show that the precision throughout the assay is adequate to size and distinguish alleles which differ by as little as one base.{Figure 4}{Figure 5}

Stutter peaks are frequently generated during the PCR process. This phenomenon makes it difficult to analyze target samples that differentiate the main allele caused by loci mutation from stutter products, especially those with a high percentage. Thus, 582 individuals were tested using the EX16+22Y system to obtain the percentage of stutter products and SD for each locus [Table 4].{Table 4}


A reproducibility study was conducted to determine if the profiles are reliable and suitable for comparison between or among laboratories with the same DNA samples. To demonstrate compatibility on different CE platforms (Applied Biosystems 3130xl and 3500xl), we run ten control DNA 9948, ten 9947A amplification products, and ten ladders on these platforms with the same POP-4™ polymer. We got consistent genotypes for the same amplification sample on each instrument. Buccal indicating FTA® Cards and blood FTA® Cards were directly amplified. Three buccal indicating FTA® Cards and three blood FTA® Cards were amplified in two laboratories, respectively. The results were consistent among all of the laboratories. Overall, the EX16+22Y system is compatible with the Applied Biosystems 3130xl and 3500xl.

Polymerase chain reaction condition study

The PCR-based conditions included testing the following reaction components: the reaction components (reaction mix, primer, and Taq polymerase), cycling number annealing temperature, and final extension time.

Reaction components

The reaction mix, primer, and Taq polymerase concentrations are important factors for consistent and robust results. The EX16+22Y system primer mix was optimized for the best balance and performance. Taq polymerase is included in the kit due to its thermal stability and demonstrated applicability for the STR multiplex. As an important cofactor for proper Taq polymerase function, magnesium is included in the reaction mix, and the concentration cannot be changed unless the reaction mix is not properly mixed or chelated. As pipetting errors may cause fluctuations in the reaction mix, primer, and Taq polymerase concentrations, 0.5 ng control DNA 9948 was amplified with 0.5×, 0.75×, 1×, 1.25×, 1.5× reaction mix, primer, and Taq polymerase [Figure 6], [Figure 7], [Figure 8]. The results showed that full profiles were obtained except at 0.5×, 1.5× reaction mix and 0.5× primer concentration. Peak height and balance were changed at 0.75×, 1.25× reaction mix and primer concentration. Variable Taq polymerase concentrations of the EX16+22Y system should not impact results significantly. Therefore, the EX16+22Y system is consistent and robust, and it tolerates various component concentrations.{Figure 6}{Figure 7}{Figure 8}

Cycle number

The recommended optimal cycling number for the EX16+22Y system is 30 cycles. Of 9948 DNA, 0.5 ng was amplified at 28, 29, 30, 31, and 32 cycles, respectively. Full profiles were reliably generated at 28 cycles or more. For all tested samples, the overall balance had no effect basically by a variation in the cycle number. As expected, the peak heights increased with additional cycles. [Figure 9] shows the mean peak heights from 1800 RFU to 8000 RFU for the control DNA 9948 amplified for different cycle numbers. Therefore, certain situations require the increased sensitivity afforded by additional cycles.{Figure 9}

Annealing temperature study

The optimized annealing temperature for the EX16+22Y system is 60°C. This annealing temperature was selected after extensive stressing of the multiplex during development and changing the annealing temperature is not recommended. However, it is possible for slight temperature variations to exist between thermal cyclers. Herein, we examined the effect of intentionally increased and decreased annealing temperatures. The appropriate annealing temperature was between 58°C and 62°C to ensure the stability and accuracy of genotyping results [Figure 10], as the precise and accurate profiles could be observed at this annealing temperature. The peak heights of DYS437 and DYS19 gradually decreased as the temperature rose after 64°C. The EX16+22Y system generated optimal results at an annealing temperature of 60°C.{Figure 10}

Final extension time

The final hold time was tested with 0 min, 10 min, 20 min (recommended), 30 min, and 40 min. As Taq polymerase has the tendency of adding an extra, on-template nucleotide at the 3′ends of DNA strands during thermal cycling.[12] Therefore, sufficient final extension time was needed to adenylate the fragments. 0.5 ng 9948 performed well at the above 20 min extension times, but the D21S11, DYS392, and FGA loci showed small minus A(−A) peaks under the condition of 0 min and 10 min and the DYS390 locus showed small plus A(+A) peaks under the condition of 30 min and 40 min. Based on these results, 20 min was recommended to be the best final extension time [Figure 11].{Figure 11}


Forensic DNA databases based on STR profiles are indispensable investigative tools for the law enforcement community. STR technologies have also been successfully applied to fight human trafficking and to identify missing persons. Their usefulness has been well publicized, resulting in the worldwide creation and expansion of forensic DNA databases.[13] To keep pace with the increasing number of reference DNA samples, databasing laboratories will need to continue to improve their efficiency and throughput.

In this article, the developmental validation of the EX16+22Y system was described. The validation studies demonstrated that the EX16+22Y system is the robust, accurate, and sensitive multiplex PCR systems which combine autosomal and Y chromosome STR loci making the system more suitable for individual identification, paternity testing, paternal lineage testing, and mixture identification. The kit is designed to accommodate direct amplification of DNA deposited on multiple substrates, so the results of the experiments demonstrated the flexibility and benefits of the kit for casework and database applications. Recently, Promega and Life Technologies already have developed commercial STR kits, respectively, PowerPlex Fusion 6C System and GlobalFiler Express PCR System, which also combine autosomal and Y chromosome STR loci. However, the Fusion and GlobalFiler include few Y chromosome STR loci, which have some limitation for mixture identification in sexual assault cases and patrilineal relationship evaluation in kinship testing. In conclusion, the EX16+22Y system is suitable for the demand of forensic DNA testing, providing a new approach for fighting against crime quickly.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


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