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An efficient novel method for analyzing STR loci from a single sperm captured by laser microdissection
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Forensic Science International: Genetics Supplement Series 1 (2008) 437–438 www.elsevier.com/locate/FSIGSS
Research article
An efficient novel method for analyzing STR loci from a single sperm captured by laser microdissection T. Miyazaki a,*, M. Hara b, A. Ichiki c, Y. Yamamoto d, A. Takada b, A. Kido e, M. Nodera a, H. Yanagisawa a, H. Suzuki a, Saito K b a
Department of Health Science & Preventive Medicine, Community Health Science Center, Japan b Department of Forensic Medicine, Faculty of Medicine, Saitama Medical University, Japan c Department of Anesthesiology, Mita Hospital, International University of Welfare & Health, Japan d Criminal Investigation Laboratory, Saitama Prefectual Police Headquarters, Japan e Department of Legal Medicine, Faculty of Medicine, University of Yamanashi, Japan Received 27 August 2007; accepted 10 October 2007
Abstract The identification of individuals using short tandem repeat (STR) analysis is important in forensic investigation. STR analysis is especially difficult using DNA from a single nucleus. In this study, DNA from a single sperm from a semen smear stained with hematoxylin and eosin (H&E) was captured using a laser microdissection method and amplified using the improved primer extension preamplification (I-PEP) polymerase chain reaction (PCR) method. Amplified DNA was used for STR analysis with the original and 3% primer methods. DNA from 15 STR loci and the amelogenin locus was amplified using an AmpFlSTR1 PCR Amplification kit (Applied Biosystems). Using the original primer method, it was impossible to genotype the 15 STR loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA). While using less primer (3% dilution), the 15 STR loci and amelogenin locus were determined perfectly in all of the preserved single sperms. Therefore, the reduced primer method is more efficient for analyzing STR loci from a single sperm captured by laser microdissection. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: STR; Single sperm; Haploid-type; Microdissection
1. Introduction
2. Materials and methods
Analysis of DNA trace samples is important for individual identification in forensic medicine, especially identification analysis from mixed or trace samples. Kane et al. [1] applied a method using less primer to multiplex polymerase chain reaction (PCR). When there is a locus-to-locus imbalance, the manufacturer recommends reducing the number of PCR cycles and amplification using less template can improve the balance among loci. We performed short tandem repeat (STR) analysis for haploid typing of single sperm captured using laser microdissection with the reduced-primer method using an AmpFlSTR Identifiler kit.
Sperms were obtained from three healthy volunteers. The samples were washed with TE buffer, and centrifuged three times at 9000 g for 5 min. The washed sperm was treated with 1.0 ml of TE buffer containing 4 mg/ml Proteinase K (QIAGEN, Hilden, Germany) at 56 8C for 30 min. The digested sample was centrifuged, and then washed five times with TE buffer to remove the Proteinase K. The treated sperm was smeared on PALMTM membrane slides (PALM Microlaser Technologies). The membrane slide was dry fixed and stained with hematoxylin and eosin (H&E). DNA from a single nucleus captured using the PALMTM Microlaser system was used for STR genotyping [2]. This DNA was used for the improved primer extension preamplification polymerase chain reaction (I-PEP-PCR), which was performed as reported [3]. PCR was carried out for 50 cycles of 94 8C for 1 min, 37 8C for 2 min, ramped to 55 8C at 0.1 8C/s, and then held at 55 8C for 4 min, and 68 8C for 30 s.
* Corresponding author. Tel.: +81 49 276 1168, fax: +81 49 294 6907. E-mail address: [email protected] (T. Miyazaki). 1875-1768/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2007.10.074
438
T. Miyazaki et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 437–438
Fig. 1. Electropherograms of STR typing by 3% primer method in single sperm. DNA of positive control was obtained from nail (A) or semen (B).
The PCR primers used were from the Power Plex1 16 Monoplex system kit (Promega, Madison, WI, USA) for the vWA and D3S1358 loci. The PCR reaction mixture consisted of 12.5 ml of Expand High Fidelity Reaction buffer No. 2 (Roche) containing 0.2 mM dNTPs, 0.5 U Expand High Fidelity Enzyme Blend, and vWA or D3S1358 primer. The PCR conditions consisted of 34 cycles of 94 8C for 1 min, 60 8C for 1 min, and 72 8C for 1 min and then a final extension at 72 8C for 60 min. An STR analysis of the DNA from a single nucleus amplified using I-PEP-PCR was performed with an AmpFlSTR Profiler PCR Amplification kit (Applied Biosystems); 16 STR loci were studied: D8S1179, D21S11, D7S820, CFS1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, FGA, and X/Y. The PCR products were separated electrophoretically on an ABI PRISM1 310 Genetic Analyzer (Applied Biosystems). The product size and genotyping were determined using GeneScanTM and GenoTyperTM, respectively.
method, the values were 54.2% or 6–12 of 16 loci, and 8.7.0 2.8 loci. The results were significantly better with the 3% primer method than with the original primer method. Using the original primer method, the STR genotyping assay was impossible for the 15 loci, while with the reduced primer method, the 15 STR loci and amelogenin locus were determined perfectly in all of the preserved single sperm. The reduced primer method allows more efficient analysis of the STR loci from a single sperm captured using laser microdissection. One of the reasons for the results with the 3% primer method may be the imbalance between template DNA and the primer concentrations.
3. Results and discussion
[1] M. Kane, S. Masui, et al., Application of less primer method to multiplex PCR, Prog. Forensic Med. 1288 (2006) 694–696. [2] Y. Yamamoto, M. Hara, et al., DNA analysis of a single sperm captured using a laser microdissection method, Res. Pract. Forensic Med. 48 (2005) 217–223. [3] Y. Yamamoto, M. Hara, et al., STR loci analysis of buccal cavity cells captured by laser microdissection, Prog. Forensic Med. 1288 (2006) 666–668.
Characteristic haploid-type electrophorograms derived from a single sperm were detected, as shown in Fig. 1(A) and (B). Using the original primer method, the STR loci type and amelogenin locus were detected for 12.5% or 0–5 of 16 loci (mean S.D.; 2.0 2.3 loci, n = 9). With the 3% primer
Conflict of interest None. References
Forensic Science International: Genetics Supplement Series 1 (2008) 437–438 www.elsevier.com/locate/FSIGSS
Research article
An efficient novel method for analyzing STR loci from a single sperm captured by laser microdissection T. Miyazaki a,*, M. Hara b, A. Ichiki c, Y. Yamamoto d, A. Takada b, A. Kido e, M. Nodera a, H. Yanagisawa a, H. Suzuki a, Saito K b a
Department of Health Science & Preventive Medicine, Community Health Science Center, Japan b Department of Forensic Medicine, Faculty of Medicine, Saitama Medical University, Japan c Department of Anesthesiology, Mita Hospital, International University of Welfare & Health, Japan d Criminal Investigation Laboratory, Saitama Prefectual Police Headquarters, Japan e Department of Legal Medicine, Faculty of Medicine, University of Yamanashi, Japan Received 27 August 2007; accepted 10 October 2007
Abstract The identification of individuals using short tandem repeat (STR) analysis is important in forensic investigation. STR analysis is especially difficult using DNA from a single nucleus. In this study, DNA from a single sperm from a semen smear stained with hematoxylin and eosin (H&E) was captured using a laser microdissection method and amplified using the improved primer extension preamplification (I-PEP) polymerase chain reaction (PCR) method. Amplified DNA was used for STR analysis with the original and 3% primer methods. DNA from 15 STR loci and the amelogenin locus was amplified using an AmpFlSTR1 PCR Amplification kit (Applied Biosystems). Using the original primer method, it was impossible to genotype the 15 STR loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA). While using less primer (3% dilution), the 15 STR loci and amelogenin locus were determined perfectly in all of the preserved single sperms. Therefore, the reduced primer method is more efficient for analyzing STR loci from a single sperm captured by laser microdissection. # 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: STR; Single sperm; Haploid-type; Microdissection
1. Introduction
2. Materials and methods
Analysis of DNA trace samples is important for individual identification in forensic medicine, especially identification analysis from mixed or trace samples. Kane et al. [1] applied a method using less primer to multiplex polymerase chain reaction (PCR). When there is a locus-to-locus imbalance, the manufacturer recommends reducing the number of PCR cycles and amplification using less template can improve the balance among loci. We performed short tandem repeat (STR) analysis for haploid typing of single sperm captured using laser microdissection with the reduced-primer method using an AmpFlSTR Identifiler kit.
Sperms were obtained from three healthy volunteers. The samples were washed with TE buffer, and centrifuged three times at 9000 g for 5 min. The washed sperm was treated with 1.0 ml of TE buffer containing 4 mg/ml Proteinase K (QIAGEN, Hilden, Germany) at 56 8C for 30 min. The digested sample was centrifuged, and then washed five times with TE buffer to remove the Proteinase K. The treated sperm was smeared on PALMTM membrane slides (PALM Microlaser Technologies). The membrane slide was dry fixed and stained with hematoxylin and eosin (H&E). DNA from a single nucleus captured using the PALMTM Microlaser system was used for STR genotyping [2]. This DNA was used for the improved primer extension preamplification polymerase chain reaction (I-PEP-PCR), which was performed as reported [3]. PCR was carried out for 50 cycles of 94 8C for 1 min, 37 8C for 2 min, ramped to 55 8C at 0.1 8C/s, and then held at 55 8C for 4 min, and 68 8C for 30 s.
* Corresponding author. Tel.: +81 49 276 1168, fax: +81 49 294 6907. E-mail address: [email protected] (T. Miyazaki). 1875-1768/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2007.10.074
438
T. Miyazaki et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 437–438
Fig. 1. Electropherograms of STR typing by 3% primer method in single sperm. DNA of positive control was obtained from nail (A) or semen (B).
The PCR primers used were from the Power Plex1 16 Monoplex system kit (Promega, Madison, WI, USA) for the vWA and D3S1358 loci. The PCR reaction mixture consisted of 12.5 ml of Expand High Fidelity Reaction buffer No. 2 (Roche) containing 0.2 mM dNTPs, 0.5 U Expand High Fidelity Enzyme Blend, and vWA or D3S1358 primer. The PCR conditions consisted of 34 cycles of 94 8C for 1 min, 60 8C for 1 min, and 72 8C for 1 min and then a final extension at 72 8C for 60 min. An STR analysis of the DNA from a single nucleus amplified using I-PEP-PCR was performed with an AmpFlSTR Profiler PCR Amplification kit (Applied Biosystems); 16 STR loci were studied: D8S1179, D21S11, D7S820, CFS1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, FGA, and X/Y. The PCR products were separated electrophoretically on an ABI PRISM1 310 Genetic Analyzer (Applied Biosystems). The product size and genotyping were determined using GeneScanTM and GenoTyperTM, respectively.
method, the values were 54.2% or 6–12 of 16 loci, and 8.7.0 2.8 loci. The results were significantly better with the 3% primer method than with the original primer method. Using the original primer method, the STR genotyping assay was impossible for the 15 loci, while with the reduced primer method, the 15 STR loci and amelogenin locus were determined perfectly in all of the preserved single sperm. The reduced primer method allows more efficient analysis of the STR loci from a single sperm captured using laser microdissection. One of the reasons for the results with the 3% primer method may be the imbalance between template DNA and the primer concentrations.
3. Results and discussion
[1] M. Kane, S. Masui, et al., Application of less primer method to multiplex PCR, Prog. Forensic Med. 1288 (2006) 694–696. [2] Y. Yamamoto, M. Hara, et al., DNA analysis of a single sperm captured using a laser microdissection method, Res. Pract. Forensic Med. 48 (2005) 217–223. [3] Y. Yamamoto, M. Hara, et al., STR loci analysis of buccal cavity cells captured by laser microdissection, Prog. Forensic Med. 1288 (2006) 666–668.
Characteristic haploid-type electrophorograms derived from a single sperm were detected, as shown in Fig. 1(A) and (B). Using the original primer method, the STR loci type and amelogenin locus were detected for 12.5% or 0–5 of 16 loci (mean S.D.; 2.0 2.3 loci, n = 9). With the 3% primer
Conflict of interest None. References