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Detection of short tandem repeat polymorphisms from human nails using direct polymerase chain reaction method
Jian Tie, Seisaku Uchigasaki
Division of Legal Medicine, Department of Social Medicine, Nihon University School of Medicine, Tokyo, Japan
Correspondence: Dr. Jian Tie, Division of Legal Medicine, Department of Social Medicine, Nihon University School of Medicine, Tokyo 173-8610, Japan E-mail: [email protected] Fax: +81-3-3972-7776
Abbreviation: STR, short tandem repeat
Keywords: Direct multiplex PCR / Forensic identification / Human fingernail / STR polymorphisms
Total number of words including references, figure legends: 2501
Received: 05-Feb-2014; Revised: 19-May-2014; Accepted: 11-Jun-2014. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/elps.201400061. This article is protected by copyright. All rights reserved.
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Abstract Human nail is an important forensic material for parental testing and individual identification in large-scale disasters. Detection of short tandem repeat (STR) polymorphism from hard tissues generally requires DNA purification, which is technically complicated and time consuming. In the present study, we attempted to detect STR polymorphisms from untreated human nail samples by direct polymerase chain reaction (PCR) amplification ®
method using the primer mixture supplied with the GenePrint SilverSTR® III System or the AmpFℓSTR® Identifiler® PCR Amplification Kit, and Tks Gflex DNA polymerase known to be effective for amplification from crude samples. A nail fragment measuring approximately 1.5 mm in breadth and 0.5 mm in length was placed directly into a PCR tube, and various PCR conditions were tested. The PCR products were analyzed by denaturing acrylamide gel electrophoresis or capillary electrophoresis. Multiple STR polymorphisms were detected successfully. This method that detects STR polymorphisms not only from fresh human fingernails, but also from old nail fragments stored at room temperature for up to 10 years is expected to become a novel DNA analytical method in forensic medicine and genetic studies.
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Short tandem repeat (STR) polymorphisms have high discrimination power, require a small quantity of sample for testing, and can be used to test old samples. Because of these advantages, STR polymorphisms have been used as the major genetic markers for DNA identification in the field of forensic medicine [1-2]. Nowadays, commercially available kits allow testing of not only autosomal STR polymorphisms but also sex chromosome STR polymorphisms, and these tools play important roles in DNA identification in forensic medicine [3-5]. Human hard tissues including nails, bones and teeth have been used as forensic materials for individual identification and parental testing. Contrary to soft tissues such as blood and body fluid stains, the hard tissues are characterized by being more resistant to DNA degradation by environmental microorganisms and ultraviolet light, thus allowing detection of STR polymorphisms even after long period of exposure outside the body [6-7]. Moreover, even if contaminated, forensic hard tissue samples can be cleansed to remove contaminants, and are therefore especially suitable for individual identification of victims in large-scale disasters and terrorist incidents. Various methods for DNA purification from hard tissues have been reported, all of which require more complicated procedures and longer time than other forensic materials [8-10]. On the other hand, the direct polymerase chain reaction (PCR) method directly amplifies DNA from untreated samples without the need for complicated DNA purification procedures, allowing simple and rapid detection of DNA polymorphisms [11-12]. The direct PCR method has been applied to the detection of DNA polymorphisms from blood stains and seminal fluid stains, and is used not only in forensic medicine but also in various other fields [13]. In the present study, we attempted for the first time to use direct PCR for detecting STR polymorphisms from human fingernails as forensic samples. Fingernail fragments approximately 1.5 mm in breadth and 5 to 11 mm in length were collected from 15 healthy volunteers. The nail samples from 10 volunteers were tested soon after collection (fresh samples), and the nail samples from 5 volunteers had been stored in individual envelops at room temperature (old samples) and tested at 1, 5 and 10 years of This article is protected by copyright. All rights reserved.
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storage. Before use, the nail samples were washed in neutral detergent to completely remove the dirt on the surface, rinsed well in distilled water, and dried. To examine the appropriate amount of nail for PCR amplification, small nail fragments measuring 0.5, 1.0 and 1.5 mm in length were cut and used in PCR. To confirm the STR polymorphisms detected from nail samples, peripheral blood was also collected from the 15 healthy volunteers. DNA was extracted from the blood using QIAamp® DNA Mini Kit (Qiagen, Chatsworth, CA, USA) and subjected to STR polymorphism analysis, as controls for the nail samples. Amplification of STR polymorphisms in nail samples by direct PCR method was ®
conducted using two PCR systems. First, the GenePrint SilverSTR® III System (Promega, Madison, WI, USA) is a multiplex PCR kit that simultaneously amplifies the STR polymorphisms of three loci: D16S539, D7S820 and D13S317. The primer mixture supplied with this kit was used. Nail fragments measuring 0.5, 1.0 and 1.5 mm in length were placed into PCR tubes as DNA templates. The reaction mixture (25 µl in total) contained 0.5 µl of Tks Gflex DNA polymerase (Takara Bio, Japan), 12.5 µl of 2 × Gflex PCR buffer (containing 2 mM Mg2+ and 400 µM dNTPs; Takara Bio), 2.5 µl of primer mixture (Promega), and 9.5 µl of nuclease-free water. Direct PCR was performed using a GeneAmp PCR system 9700 thermal cycler (Applied Biosystems, Foster City, USA). Two PCR amplification conditions were tested (Table 1A). For STR genotyping, the PCR products were subjected to allele analysis using a 6.5% denaturing acrylamide gel containing 7 M uric acid. PCR product (2.3 µl) was mixed with an equal volume of loading solution, heat denatured at 95ºC for 3 min, and applied to the gel. Electrophoresis was conducted at constant voltage of 750 volt for 2 h. After electrophoresis, the gel was DNA stained with SYBR Green I (Sigma®, Saint Louis, MO, USA). Molecular Imager® Gel DocTM XR (BIO-RAD, Berkeley, CA, USA) was then used for detection and photography of STR polymorphisms. Second, the AmpFℓSTR® Identifiler® PCR Amplification Kit (Applied Biosystems) is a multiplex PCR system that simultaneously amplifies 15 STRs: D8S1179, D21S11, D7S820, CSFOP1, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA, plus the amelogenin locus. The primer mixture supplied with this kit was used. Nail fragments measuring 0.5 mm This article is protected by copyright. All rights reserved.
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in length were placed into PCR tubes. The reaction mixture (25 µl in total) contained 0.5 µl of Tks Gflex DNA polymerase, 12.5 µl of 2 × Gflex PCR buffer (Takara Bio), 5 µl of primer mixture (Applied Biosystems) and 7 µl of nuclease-free water. Three PCR amplification conditions were tested (Table 1B). For STR genotyping, the PCR products were subjected to allele analysis at each locus by capillary electrophoresis using the ABI PRISM 310 Genetic Analyzer, according to the manual of the kit. Genotypes were identified using GeneMapperID ®
v3.2 software. K562 DNA supplied with the GenePrint System and 9947A DNA supplied with the AmpFℓSTR® Kit were used as DNA standards. All samples were tested in duplicate. Condition 1 in Table 1A shows the PCR thermocycling conditions recommended in the manual of the GenePrint
®
System, with slight modification. While the manufacturer
recommended 20 cycles in the second cycling for testing purified DNA template, we increased the second cycling to 30 cycles to detect STR polymorphisms from nail samples by direct PCR. Using this amplification condition, in addition to the bands that should be found at given loci, extra bands were amplified (Figure 1A). Therefore, accurate genotyping was not possible. However, when PCR condition 2 was used to amplify the same samples, there were no extra bands, and sharp bands were amplified. Using this PCR protocol, all the allelic main bands were detected clearly from the nail samples of all 15 subjects, and genotyping of each locus was possible. Compared to the control DNA purified from blood of each subject, no conflicting genotypes for the three STR loci were observed (Table 2). When nail samples of three different lengths were analyzed, STR polymorphisms were amplified and genotypes could be identified in all samples, irrespective of the sample size (Figure 1B). Figure 1C shows the results of STR polymorphisms obtained from old nails of 5 subjects, which had been stored at room temperature for 1, 5 and 10 years. All genotypes were identified from nails that had been preserved for 10 years, and no difference in STR polymorphism was observed when compared with more recent samples. Next, we attempted to use capillary electrophoresis to detect 16 loci simultaneously from one PCR, when nail samples were amplified by direct PCR under three conditions (Table 1B).
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The PCR thermocycling conditions for purified DNA recommended in the manual of the AmpFℓSTR® Kit (condition 1) did not amplify all STR polymorphisms from nail samples. When we increased the time of initial incubation to 95ºC and increased the temperature and time of the denaturation step (condition 3), some peaks that were not detected by condition 1 became detectable. Eventually, all the allele peaks were detected from the nail samples of all 15 subjects. Compared to the STR polymorphisms obtained from control DNA, all the polymorphisms were accurately detected (Figure 1D). In all samples, the STRs at the three ®
loci of D16S539, D7S820 and D13S317 detected using the GenePrint SilverSTR® III System were found to have the same genotypes as those detected using the AmpFℓSTR® Identifiler® PCR Amplification Kit (Table 2). Human nail, bone, tooth and hair are important hard tissues for forensic DNA analysis. Hard tissues have advantages over soft tissues in that DNA identification is possible even after a relatively long storage period [14], and PCR inhibitors are less likely to be produced. Thus hard tissues are used in both forensic and clinical medicine [15, 16]. The direct PCR method is simple and easy for DNA polymorphism analysis, without loss of DNA by pretreatment processes, and utilization in forensic medicine is anticipated [17, 18]. Although DNA polymorphism analyses by direct PCR method have been reported for bloodstain, saliva, sperm and hair root samples, there is no report for hard tissues such as nails [19]. With recent technological improvements in PCR amplification, various DNA polymerases that are effective for PCR amplification from crude samples or samples difficult to amplify by conventional methods have been developed [20,21]. An example of such DNA polymerase is the Tks GflexTM DNA polymerase used in the present study. Using this polymerase with two commercial PCR kits and analyzed by both conventional denaturing acrylamide gel electrophoresis and the highly sensitive capillary electrophoresis, we attempted for the first time to detect STR polymorphisms from untreated nail samples by direct PCR amplification. ®
In the GenePrint SilverSTR® III System, the bands of alleles tended to be denser as the quantity of nail sample increased. However, this phenomenon was not observed with the This article is protected by copyright. All rights reserved.
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AmpFℓSTR® Identifiler® PCR Amplification Kit. For both methods, STR polymorphisms were detected when nail samples measuring 1.5 mm in breadth and 0.5 mm in length were used. Conventionally, approximately 10 mg of nail fragment is required to test STR polymorphisms because of the need to purify DNA [22]. In the present study, the maximum weight of nail used was less than 0.5 mg. In situations involving large numbers of victims, such as large-scale disasters, accidents and terrorist incidents, environmental or seasonal factors may hamper the collection and preservation of blood and soft tissues. Under such circumstances, human nail is a suitable material for individual identification of victims. DNA profiling and matching is a primary method used to identify missing persons in disasters. Together with odontology and dactyloscopy, the use of DNA is recommended by the Interpol guidelines [23]. DNA typing has played a more prominent role in the identification of human remains, and DNA sample collection and DNA-based victim identification strategy have become more important. The biggest merits of the present method are that a small quantity of nail suffices and that STR polymorphism testing is further simplified. In conclusion, using human nail samples of approximately 0.5 mm in length, multiple STR polymorphisms can be detected using denaturing acrylamide gel electrophoresis or capillary electrophoresis by direct PCR method without the need for DNA extraction. Compared to the conventional method, the direct PCR method is simple and avoids the risk of contamination due to DNA extraction and interference of PCR by extraction reagents.
Acknowledgements
This work was supported by Japan Society for the Promotion of Science KAKENHI, Great-in-Aid for Scientific Research (C) number 24590867.
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References [1] Gaibar, M., Esteban, M. E., Via, M., Harich, N., Kandil, M., Fernández-Santander, A., Ann.
Hum. Biol. 2012, 39, 297-304. [2]
de
Lourdes
Chávez-Briones,
M.
L.,
Hernández-Cortés,
R.,
Díaz-Torres,
P.,
Niderhauser-García, A., Ancer-Rodríguez, J., Jaramillo-Rangel, G., Ortega-Martínez, M.,
J Forensic Sci. 2013, 58, 248-250. [3] Harder, M., Renneberg, R., Meyer, P., Krause-Kyora, B., von Wurmb-Schwark, N., Croat.
Med. J. 2012, 53, 416-422. [4] Uchigasaki, S., Tie, J., Takahashi, D., Mol. Biol. Rep. 2013, 40, 3193-3196. [5] Davis, C., Ge, J., Sprecher, C., Chidambaram, A., Thompson, J., Ewing, M., Fulmer, P., Rabbach, D., Storts, D., Budowle, B., Forensic Sci. Int. Genet. 2013, 7, 204-208. [6] Adler, C. J., Haak, W., Donlon, D., Cooper A., J. Arch. Sci. 2011, 38, 956–964. [7] Piccinini, A., Cucurachi, N., Betti, F., Capra, M., Coco, S., D'Avila, F., Lorenzoni, R., Lovisolo, A., Int. Cong. Ser., 2006, 1288, 586–588. [8] Campos, P.F., Craig, O.E., Turner-Walker, G., Peacock, E., Willerslev, E., Gilbert, M.T.,
Ann. Anat. 2012, 194, 7-16. [9] Higgins, D., Austin, J. J., Sci. Just. 2013, 53, 433-441. [10] Allouche, M., Hamdoum, M., Mangin, P., Castella, V., Forensic Sci. Int. Genet. 2008, 3, 46-49. [11] Sim, J. E., Park, S. J., Lee, H. C., Kim, S. Y., Kim, J. Y., Lee, S. H., J. Forensic Sci. 2013,
58, 989-992. [12] Aboud, M., Oh, H. H., McCord, B., Electrophoresis, 2013, 34, 1539-1547. [13] Tie, J., Uchigasaki, S., Hasaba, T., Ohno, Y., Isahai, I., Oshida, S., Electrophoresis, 2010,
31, 2411-2415. [14] Nakanishi, A., Moriya, F., Hashimoto, Y., Leg. Med. 2003, 5, S194-S197. [15] Uchida, K., Wang, L., Yahagi, Y., Tokunaga, K., Tadokoro, K., Juji, T., Blood. 1996, 87, 4015-4016. This article is protected by copyright. All rights reserved.
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[16] Barta, J. L., Monroe, C., Kemp, B. M., Forensic Sci. Int. 2013, 231, 340-348. [17] Linacre, A., Pekarek, V., Swaran, Y. C., Tobe, S. S., Forensic Sci. Int. Genet. 2010, 4, 137-141. [18] Swaran, Y. C., Welch, L., Forensic Sci. Int. Genet. 2012, 6, 407-412. [19] Verheij, S., Harteveld, J., Sijen, T., Forensic Sci. Int. Genet. 2012, 6, 167-175. [20] Quinceya, D., Carlea, G., Alunnib, V., Quatrehommeb, G., Sci. Justice, 2013, 53, 253–260. [21] Purzycka, J.K., Olewiecki, I., Soltyszewski, I., Pepinski, W., Janica, J., Int. Congr. Ser. 2006, 1288, 719-721. [22] Park, J., Liang, D., Kim, J. W., Luo, Y., Huang, T., Kim, S. Y., Chang, S. S., J. Prev. Med.
Public Health. 2012, 45, 235-243. [23] Interpol, Disaster Victim Identification. http//www.interpol. int/Public/Disaster Victim/Guide/Guide.pdf.
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Figure 1. A to C: Polyacrylamide gel electrophoresis images showing STR polymorphisms ® amplified from nail samples by direct PCR using the GenePrint SilverSTR® III System. A: L is the allelic ladders. Lanes 1-4 were amplified using PCR condition 1, and lanes 5-8 using PCR condition 2 (for details, see Table 1A). Lanes 1 and 5, lanes 2 and 6, lanes 3 and 7, and lanes 4 and 8 are the same samples. B: Lanes 1-3, lanes 4-6, and lanes 7-9 are the same samples of various lengths; 0.5 mm (lanes 1, 4 and 7), 1.0 mm (lanes 2, 5 and 8) and 1.5 mm (lanes 3, 6 and 9). C: Lanes 1-3, lanes 4-6, and lanes 7-9 are the same samples stored at room temperature for 1 year (lanes 1, 4 and 7), 5 years (lanes 2, 5 and 8) and 10 years (lanes 3, 6 and 9). D: Capillary electropherogram showing STR polymorphisms amplified from nail samples by direct PCR using the AmpFℓSTR® Identifiler® PCR Amplification Kit. 1, 2 and 3 show the D3S1358, TH01, D13S317, D16S539 and D2S1338 loci amplified by PCR cycling conditions 1, 2 and 3, respectively (for details, see Table 1B). The nail sample is the same sample used in lanes 4-6 of Figure 1B, and the genotypes detected at D13S317 and D16S539 loci are identical to those obtained in Figure 1B. Figure.1
L 1 2 3 4
5 6
7 8 L D16S539
a
D7S820
D13S317
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L 1 2 3 4
5 6
Electrophoresis
7 8
9 L D16S539
b
D7S820
D13S317
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L 1 2 3 4
c
5 6
Electrophoresis
7 8
9 L D16S539
D7S820
D13S317
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Table 1. Amplification conditions for human nails by direct PCR using two commercial PCR systems. ® A. GenePrint SilverSTR® III System Protocol Initial First cycling: Second cycling: Extension Hold incubation 10 cycles 30 cycles step step 96ºC 94ºC, 30 seconds 90ºC, 30 seconds 60ºC, 4ºC 1 60ºC, 30 seconds 60ºC, 30 seconds 1 minute 30 minutes 70ºC, 45 seconds 70ºC, 45 seconds 96ºC 15 minutes
2
98ºC, 15 seconds 60ºC, 30 seconds 70ºC, 1 minute
95ºC, 15 seconds 60ºC, 30 seconds 70ºC, 1 minute
B. AmpFℓSTR® Identifiler® PCR Amplification Kit Protocol Initial Cycling (37 cycles) incubation Denaturation Annealing
60ºC, 30 minutes
4ºC
Hold Extension
1
95ºC for 11 minutes
94ºC, for 1.0 minute
59ºC, for 1.0 minute
72ºC, for 1.0 minute
25ºC
2
95ºC for 15 minutes
95ºC, for 1.0 minute
59ºC, for 1.0 minute
72ºC, for 1.0 minute
25ºC
3
95ºC for 15 minutes
96ºC, for 1.5 minutes
59ºC, for 1.0 minute
72ºC, for 1.0 minute
25ºC
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Table 2. Comparison of STR genotypes obtained from direct PCR of the nails with the genotypes detected from blood DNA in 15 individuals, for two PCR systems. Sample No.
GenePrint® SilverSTR®IIIPCR Nail Blood DNA D16S539
D7S820
D13S317
D16S539
D7S820
D13S317
AmpFℓSTR® Identifiler® PCR Nail Blood DNA D16S539
D7S820
D13S317
D16S539
D7S820
D13S317
9-12 12-12 10-12 9-12 12-12 10-12 9-12 12-12 10-12 9-12 12-12 10-12 1 9-9 9-10 8-11 9-9 9-10 8-11 9-9 9-10 8-11 9-9 9-10 8-11 2 11-13 10-13 8-11 11-13 10-13 8-11 11-13 10-13 8-11 11-13 10-13 8-11 3 10-11 10-12 9-13 10-11 10-12 9-13 10-11 10-12 9-13 10-11 10-12 9-13 4 10-10 8-13 8-9 10-10 8-13 8-9 10-10 8-13 8-9 10-10 8-13 8-9 5* 10-11 10-12 10-12 10-11 10-12 10-12 10-11 10-12 10-12 10-11 10-12 10-12 6 9-14 10-12 10-12 9-14 10-12 10-12 9-14 10-12 10-12 9-14 10-12 10-12 7 9-12 10-11 10-12 9-12 10-11 10-12 9-12 10-11 10-12 9-12 10-11 10-12 8 12-12 12-12 10-12 12-12 12-12 10-12 12-12 12-12 10-12 12-12 12-12 10-12 9 8-11 9-10 7-10 8-11 9-10 7-10 8-11 9-10 7-10 8-11 9-10 7-10 10 8-9 7-10 7-12 8-9 7-10 7-12 8-9 7-10 7-12 8-9 7-10 7-12 11 9-10 10-12 8-10 9-10 10-12 8-10 9-10 10-12 8-10 9-10 10-12 8-10 12 9-9 10-11 9-11 9-9 10-11 9-11 9-9 10-11 9-11 9-9 10-11 9-11 13 9-10 11-11 11-13 9-10 11-11 11-13 9-10 11-11 11-13 9-10 11-11 11-13 14 10-11 11-11 8-8 10-11 11-11 8-8 10-11 11-11 8-8 10-11 11-11 8-8 15 *The electrophoresis results of this sample are shown in lanes 4-6 of Figure 1B and in Figure 1D for D13S317 and D16S539 loci.
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Detection of short tandem repeat polymorphisms from human nails using direct polymerase chain reaction method
Jian Tie, Seisaku Uchigasaki
Division of Legal Medicine, Department of Social Medicine, Nihon University School of Medicine, Tokyo, Japan
Correspondence: Dr. Jian Tie, Division of Legal Medicine, Department of Social Medicine, Nihon University School of Medicine, Tokyo 173-8610, Japan E-mail: [email protected] Fax: +81-3-3972-7776
Abbreviation: STR, short tandem repeat
Keywords: Direct multiplex PCR / Forensic identification / Human fingernail / STR polymorphisms
Total number of words including references, figure legends: 2501
Received: 05-Feb-2014; Revised: 19-May-2014; Accepted: 11-Jun-2014. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/elps.201400061. This article is protected by copyright. All rights reserved.
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Abstract Human nail is an important forensic material for parental testing and individual identification in large-scale disasters. Detection of short tandem repeat (STR) polymorphism from hard tissues generally requires DNA purification, which is technically complicated and time consuming. In the present study, we attempted to detect STR polymorphisms from untreated human nail samples by direct polymerase chain reaction (PCR) amplification ®
method using the primer mixture supplied with the GenePrint SilverSTR® III System or the AmpFℓSTR® Identifiler® PCR Amplification Kit, and Tks Gflex DNA polymerase known to be effective for amplification from crude samples. A nail fragment measuring approximately 1.5 mm in breadth and 0.5 mm in length was placed directly into a PCR tube, and various PCR conditions were tested. The PCR products were analyzed by denaturing acrylamide gel electrophoresis or capillary electrophoresis. Multiple STR polymorphisms were detected successfully. This method that detects STR polymorphisms not only from fresh human fingernails, but also from old nail fragments stored at room temperature for up to 10 years is expected to become a novel DNA analytical method in forensic medicine and genetic studies.
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Short tandem repeat (STR) polymorphisms have high discrimination power, require a small quantity of sample for testing, and can be used to test old samples. Because of these advantages, STR polymorphisms have been used as the major genetic markers for DNA identification in the field of forensic medicine [1-2]. Nowadays, commercially available kits allow testing of not only autosomal STR polymorphisms but also sex chromosome STR polymorphisms, and these tools play important roles in DNA identification in forensic medicine [3-5]. Human hard tissues including nails, bones and teeth have been used as forensic materials for individual identification and parental testing. Contrary to soft tissues such as blood and body fluid stains, the hard tissues are characterized by being more resistant to DNA degradation by environmental microorganisms and ultraviolet light, thus allowing detection of STR polymorphisms even after long period of exposure outside the body [6-7]. Moreover, even if contaminated, forensic hard tissue samples can be cleansed to remove contaminants, and are therefore especially suitable for individual identification of victims in large-scale disasters and terrorist incidents. Various methods for DNA purification from hard tissues have been reported, all of which require more complicated procedures and longer time than other forensic materials [8-10]. On the other hand, the direct polymerase chain reaction (PCR) method directly amplifies DNA from untreated samples without the need for complicated DNA purification procedures, allowing simple and rapid detection of DNA polymorphisms [11-12]. The direct PCR method has been applied to the detection of DNA polymorphisms from blood stains and seminal fluid stains, and is used not only in forensic medicine but also in various other fields [13]. In the present study, we attempted for the first time to use direct PCR for detecting STR polymorphisms from human fingernails as forensic samples. Fingernail fragments approximately 1.5 mm in breadth and 5 to 11 mm in length were collected from 15 healthy volunteers. The nail samples from 10 volunteers were tested soon after collection (fresh samples), and the nail samples from 5 volunteers had been stored in individual envelops at room temperature (old samples) and tested at 1, 5 and 10 years of This article is protected by copyright. All rights reserved.
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storage. Before use, the nail samples were washed in neutral detergent to completely remove the dirt on the surface, rinsed well in distilled water, and dried. To examine the appropriate amount of nail for PCR amplification, small nail fragments measuring 0.5, 1.0 and 1.5 mm in length were cut and used in PCR. To confirm the STR polymorphisms detected from nail samples, peripheral blood was also collected from the 15 healthy volunteers. DNA was extracted from the blood using QIAamp® DNA Mini Kit (Qiagen, Chatsworth, CA, USA) and subjected to STR polymorphism analysis, as controls for the nail samples. Amplification of STR polymorphisms in nail samples by direct PCR method was ®
conducted using two PCR systems. First, the GenePrint SilverSTR® III System (Promega, Madison, WI, USA) is a multiplex PCR kit that simultaneously amplifies the STR polymorphisms of three loci: D16S539, D7S820 and D13S317. The primer mixture supplied with this kit was used. Nail fragments measuring 0.5, 1.0 and 1.5 mm in length were placed into PCR tubes as DNA templates. The reaction mixture (25 µl in total) contained 0.5 µl of Tks Gflex DNA polymerase (Takara Bio, Japan), 12.5 µl of 2 × Gflex PCR buffer (containing 2 mM Mg2+ and 400 µM dNTPs; Takara Bio), 2.5 µl of primer mixture (Promega), and 9.5 µl of nuclease-free water. Direct PCR was performed using a GeneAmp PCR system 9700 thermal cycler (Applied Biosystems, Foster City, USA). Two PCR amplification conditions were tested (Table 1A). For STR genotyping, the PCR products were subjected to allele analysis using a 6.5% denaturing acrylamide gel containing 7 M uric acid. PCR product (2.3 µl) was mixed with an equal volume of loading solution, heat denatured at 95ºC for 3 min, and applied to the gel. Electrophoresis was conducted at constant voltage of 750 volt for 2 h. After electrophoresis, the gel was DNA stained with SYBR Green I (Sigma®, Saint Louis, MO, USA). Molecular Imager® Gel DocTM XR (BIO-RAD, Berkeley, CA, USA) was then used for detection and photography of STR polymorphisms. Second, the AmpFℓSTR® Identifiler® PCR Amplification Kit (Applied Biosystems) is a multiplex PCR system that simultaneously amplifies 15 STRs: D8S1179, D21S11, D7S820, CSFOP1, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA, plus the amelogenin locus. The primer mixture supplied with this kit was used. Nail fragments measuring 0.5 mm This article is protected by copyright. All rights reserved.
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in length were placed into PCR tubes. The reaction mixture (25 µl in total) contained 0.5 µl of Tks Gflex DNA polymerase, 12.5 µl of 2 × Gflex PCR buffer (Takara Bio), 5 µl of primer mixture (Applied Biosystems) and 7 µl of nuclease-free water. Three PCR amplification conditions were tested (Table 1B). For STR genotyping, the PCR products were subjected to allele analysis at each locus by capillary electrophoresis using the ABI PRISM 310 Genetic Analyzer, according to the manual of the kit. Genotypes were identified using GeneMapperID ®
v3.2 software. K562 DNA supplied with the GenePrint System and 9947A DNA supplied with the AmpFℓSTR® Kit were used as DNA standards. All samples were tested in duplicate. Condition 1 in Table 1A shows the PCR thermocycling conditions recommended in the manual of the GenePrint
®
System, with slight modification. While the manufacturer
recommended 20 cycles in the second cycling for testing purified DNA template, we increased the second cycling to 30 cycles to detect STR polymorphisms from nail samples by direct PCR. Using this amplification condition, in addition to the bands that should be found at given loci, extra bands were amplified (Figure 1A). Therefore, accurate genotyping was not possible. However, when PCR condition 2 was used to amplify the same samples, there were no extra bands, and sharp bands were amplified. Using this PCR protocol, all the allelic main bands were detected clearly from the nail samples of all 15 subjects, and genotyping of each locus was possible. Compared to the control DNA purified from blood of each subject, no conflicting genotypes for the three STR loci were observed (Table 2). When nail samples of three different lengths were analyzed, STR polymorphisms were amplified and genotypes could be identified in all samples, irrespective of the sample size (Figure 1B). Figure 1C shows the results of STR polymorphisms obtained from old nails of 5 subjects, which had been stored at room temperature for 1, 5 and 10 years. All genotypes were identified from nails that had been preserved for 10 years, and no difference in STR polymorphism was observed when compared with more recent samples. Next, we attempted to use capillary electrophoresis to detect 16 loci simultaneously from one PCR, when nail samples were amplified by direct PCR under three conditions (Table 1B).
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The PCR thermocycling conditions for purified DNA recommended in the manual of the AmpFℓSTR® Kit (condition 1) did not amplify all STR polymorphisms from nail samples. When we increased the time of initial incubation to 95ºC and increased the temperature and time of the denaturation step (condition 3), some peaks that were not detected by condition 1 became detectable. Eventually, all the allele peaks were detected from the nail samples of all 15 subjects. Compared to the STR polymorphisms obtained from control DNA, all the polymorphisms were accurately detected (Figure 1D). In all samples, the STRs at the three ®
loci of D16S539, D7S820 and D13S317 detected using the GenePrint SilverSTR® III System were found to have the same genotypes as those detected using the AmpFℓSTR® Identifiler® PCR Amplification Kit (Table 2). Human nail, bone, tooth and hair are important hard tissues for forensic DNA analysis. Hard tissues have advantages over soft tissues in that DNA identification is possible even after a relatively long storage period [14], and PCR inhibitors are less likely to be produced. Thus hard tissues are used in both forensic and clinical medicine [15, 16]. The direct PCR method is simple and easy for DNA polymorphism analysis, without loss of DNA by pretreatment processes, and utilization in forensic medicine is anticipated [17, 18]. Although DNA polymorphism analyses by direct PCR method have been reported for bloodstain, saliva, sperm and hair root samples, there is no report for hard tissues such as nails [19]. With recent technological improvements in PCR amplification, various DNA polymerases that are effective for PCR amplification from crude samples or samples difficult to amplify by conventional methods have been developed [20,21]. An example of such DNA polymerase is the Tks GflexTM DNA polymerase used in the present study. Using this polymerase with two commercial PCR kits and analyzed by both conventional denaturing acrylamide gel electrophoresis and the highly sensitive capillary electrophoresis, we attempted for the first time to detect STR polymorphisms from untreated nail samples by direct PCR amplification. ®
In the GenePrint SilverSTR® III System, the bands of alleles tended to be denser as the quantity of nail sample increased. However, this phenomenon was not observed with the This article is protected by copyright. All rights reserved.
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AmpFℓSTR® Identifiler® PCR Amplification Kit. For both methods, STR polymorphisms were detected when nail samples measuring 1.5 mm in breadth and 0.5 mm in length were used. Conventionally, approximately 10 mg of nail fragment is required to test STR polymorphisms because of the need to purify DNA [22]. In the present study, the maximum weight of nail used was less than 0.5 mg. In situations involving large numbers of victims, such as large-scale disasters, accidents and terrorist incidents, environmental or seasonal factors may hamper the collection and preservation of blood and soft tissues. Under such circumstances, human nail is a suitable material for individual identification of victims. DNA profiling and matching is a primary method used to identify missing persons in disasters. Together with odontology and dactyloscopy, the use of DNA is recommended by the Interpol guidelines [23]. DNA typing has played a more prominent role in the identification of human remains, and DNA sample collection and DNA-based victim identification strategy have become more important. The biggest merits of the present method are that a small quantity of nail suffices and that STR polymorphism testing is further simplified. In conclusion, using human nail samples of approximately 0.5 mm in length, multiple STR polymorphisms can be detected using denaturing acrylamide gel electrophoresis or capillary electrophoresis by direct PCR method without the need for DNA extraction. Compared to the conventional method, the direct PCR method is simple and avoids the risk of contamination due to DNA extraction and interference of PCR by extraction reagents.
Acknowledgements
This work was supported by Japan Society for the Promotion of Science KAKENHI, Great-in-Aid for Scientific Research (C) number 24590867.
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References [1] Gaibar, M., Esteban, M. E., Via, M., Harich, N., Kandil, M., Fernández-Santander, A., Ann.
Hum. Biol. 2012, 39, 297-304. [2]
de
Lourdes
Chávez-Briones,
M.
L.,
Hernández-Cortés,
R.,
Díaz-Torres,
P.,
Niderhauser-García, A., Ancer-Rodríguez, J., Jaramillo-Rangel, G., Ortega-Martínez, M.,
J Forensic Sci. 2013, 58, 248-250. [3] Harder, M., Renneberg, R., Meyer, P., Krause-Kyora, B., von Wurmb-Schwark, N., Croat.
Med. J. 2012, 53, 416-422. [4] Uchigasaki, S., Tie, J., Takahashi, D., Mol. Biol. Rep. 2013, 40, 3193-3196. [5] Davis, C., Ge, J., Sprecher, C., Chidambaram, A., Thompson, J., Ewing, M., Fulmer, P., Rabbach, D., Storts, D., Budowle, B., Forensic Sci. Int. Genet. 2013, 7, 204-208. [6] Adler, C. J., Haak, W., Donlon, D., Cooper A., J. Arch. Sci. 2011, 38, 956–964. [7] Piccinini, A., Cucurachi, N., Betti, F., Capra, M., Coco, S., D'Avila, F., Lorenzoni, R., Lovisolo, A., Int. Cong. Ser., 2006, 1288, 586–588. [8] Campos, P.F., Craig, O.E., Turner-Walker, G., Peacock, E., Willerslev, E., Gilbert, M.T.,
Ann. Anat. 2012, 194, 7-16. [9] Higgins, D., Austin, J. J., Sci. Just. 2013, 53, 433-441. [10] Allouche, M., Hamdoum, M., Mangin, P., Castella, V., Forensic Sci. Int. Genet. 2008, 3, 46-49. [11] Sim, J. E., Park, S. J., Lee, H. C., Kim, S. Y., Kim, J. Y., Lee, S. H., J. Forensic Sci. 2013,
58, 989-992. [12] Aboud, M., Oh, H. H., McCord, B., Electrophoresis, 2013, 34, 1539-1547. [13] Tie, J., Uchigasaki, S., Hasaba, T., Ohno, Y., Isahai, I., Oshida, S., Electrophoresis, 2010,
31, 2411-2415. [14] Nakanishi, A., Moriya, F., Hashimoto, Y., Leg. Med. 2003, 5, S194-S197. [15] Uchida, K., Wang, L., Yahagi, Y., Tokunaga, K., Tadokoro, K., Juji, T., Blood. 1996, 87, 4015-4016. This article is protected by copyright. All rights reserved.
www.electrophoresis-journal.com
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[16] Barta, J. L., Monroe, C., Kemp, B. M., Forensic Sci. Int. 2013, 231, 340-348. [17] Linacre, A., Pekarek, V., Swaran, Y. C., Tobe, S. S., Forensic Sci. Int. Genet. 2010, 4, 137-141. [18] Swaran, Y. C., Welch, L., Forensic Sci. Int. Genet. 2012, 6, 407-412. [19] Verheij, S., Harteveld, J., Sijen, T., Forensic Sci. Int. Genet. 2012, 6, 167-175. [20] Quinceya, D., Carlea, G., Alunnib, V., Quatrehommeb, G., Sci. Justice, 2013, 53, 253–260. [21] Purzycka, J.K., Olewiecki, I., Soltyszewski, I., Pepinski, W., Janica, J., Int. Congr. Ser. 2006, 1288, 719-721. [22] Park, J., Liang, D., Kim, J. W., Luo, Y., Huang, T., Kim, S. Y., Chang, S. S., J. Prev. Med.
Public Health. 2012, 45, 235-243. [23] Interpol, Disaster Victim Identification. http//www.interpol. int/Public/Disaster Victim/Guide/Guide.pdf.
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Figure 1. A to C: Polyacrylamide gel electrophoresis images showing STR polymorphisms ® amplified from nail samples by direct PCR using the GenePrint SilverSTR® III System. A: L is the allelic ladders. Lanes 1-4 were amplified using PCR condition 1, and lanes 5-8 using PCR condition 2 (for details, see Table 1A). Lanes 1 and 5, lanes 2 and 6, lanes 3 and 7, and lanes 4 and 8 are the same samples. B: Lanes 1-3, lanes 4-6, and lanes 7-9 are the same samples of various lengths; 0.5 mm (lanes 1, 4 and 7), 1.0 mm (lanes 2, 5 and 8) and 1.5 mm (lanes 3, 6 and 9). C: Lanes 1-3, lanes 4-6, and lanes 7-9 are the same samples stored at room temperature for 1 year (lanes 1, 4 and 7), 5 years (lanes 2, 5 and 8) and 10 years (lanes 3, 6 and 9). D: Capillary electropherogram showing STR polymorphisms amplified from nail samples by direct PCR using the AmpFℓSTR® Identifiler® PCR Amplification Kit. 1, 2 and 3 show the D3S1358, TH01, D13S317, D16S539 and D2S1338 loci amplified by PCR cycling conditions 1, 2 and 3, respectively (for details, see Table 1B). The nail sample is the same sample used in lanes 4-6 of Figure 1B, and the genotypes detected at D13S317 and D16S539 loci are identical to those obtained in Figure 1B. Figure.1
L 1 2 3 4
5 6
7 8 L D16S539
a
D7S820
D13S317
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L 1 2 3 4
5 6
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7 8
9 L D16S539
b
D7S820
D13S317
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L 1 2 3 4
c
5 6
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7 8
9 L D16S539
D7S820
D13S317
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Table 1. Amplification conditions for human nails by direct PCR using two commercial PCR systems. ® A. GenePrint SilverSTR® III System Protocol Initial First cycling: Second cycling: Extension Hold incubation 10 cycles 30 cycles step step 96ºC 94ºC, 30 seconds 90ºC, 30 seconds 60ºC, 4ºC 1 60ºC, 30 seconds 60ºC, 30 seconds 1 minute 30 minutes 70ºC, 45 seconds 70ºC, 45 seconds 96ºC 15 minutes
2
98ºC, 15 seconds 60ºC, 30 seconds 70ºC, 1 minute
95ºC, 15 seconds 60ºC, 30 seconds 70ºC, 1 minute
B. AmpFℓSTR® Identifiler® PCR Amplification Kit Protocol Initial Cycling (37 cycles) incubation Denaturation Annealing
60ºC, 30 minutes
4ºC
Hold Extension
1
95ºC for 11 minutes
94ºC, for 1.0 minute
59ºC, for 1.0 minute
72ºC, for 1.0 minute
25ºC
2
95ºC for 15 minutes
95ºC, for 1.0 minute
59ºC, for 1.0 minute
72ºC, for 1.0 minute
25ºC
3
95ºC for 15 minutes
96ºC, for 1.5 minutes
59ºC, for 1.0 minute
72ºC, for 1.0 minute
25ºC
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Table 2. Comparison of STR genotypes obtained from direct PCR of the nails with the genotypes detected from blood DNA in 15 individuals, for two PCR systems. Sample No.
GenePrint® SilverSTR®IIIPCR Nail Blood DNA D16S539
D7S820
D13S317
D16S539
D7S820
D13S317
AmpFℓSTR® Identifiler® PCR Nail Blood DNA D16S539
D7S820
D13S317
D16S539
D7S820
D13S317
9-12 12-12 10-12 9-12 12-12 10-12 9-12 12-12 10-12 9-12 12-12 10-12 1 9-9 9-10 8-11 9-9 9-10 8-11 9-9 9-10 8-11 9-9 9-10 8-11 2 11-13 10-13 8-11 11-13 10-13 8-11 11-13 10-13 8-11 11-13 10-13 8-11 3 10-11 10-12 9-13 10-11 10-12 9-13 10-11 10-12 9-13 10-11 10-12 9-13 4 10-10 8-13 8-9 10-10 8-13 8-9 10-10 8-13 8-9 10-10 8-13 8-9 5* 10-11 10-12 10-12 10-11 10-12 10-12 10-11 10-12 10-12 10-11 10-12 10-12 6 9-14 10-12 10-12 9-14 10-12 10-12 9-14 10-12 10-12 9-14 10-12 10-12 7 9-12 10-11 10-12 9-12 10-11 10-12 9-12 10-11 10-12 9-12 10-11 10-12 8 12-12 12-12 10-12 12-12 12-12 10-12 12-12 12-12 10-12 12-12 12-12 10-12 9 8-11 9-10 7-10 8-11 9-10 7-10 8-11 9-10 7-10 8-11 9-10 7-10 10 8-9 7-10 7-12 8-9 7-10 7-12 8-9 7-10 7-12 8-9 7-10 7-12 11 9-10 10-12 8-10 9-10 10-12 8-10 9-10 10-12 8-10 9-10 10-12 8-10 12 9-9 10-11 9-11 9-9 10-11 9-11 9-9 10-11 9-11 9-9 10-11 9-11 13 9-10 11-11 11-13 9-10 11-11 11-13 9-10 11-11 11-13 9-10 11-11 11-13 14 10-11 11-11 8-8 10-11 11-11 8-8 10-11 11-11 8-8 10-11 11-11 8-8 15 *The electrophoresis results of this sample are shown in lanes 4-6 of Figure 1B and in Figure 1D for D13S317 and D16S539 loci.
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