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The combination of single cell micromanipulation with LV-PCR system and its application in forensic science
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Forensic Science International: Genetics Supplement Series 2 (2009) 516–517 www.elsevier.com/locate/FSIGSS
Research article
The combination of single cell micromanipulation with LV-PCR system and its application in forensic science Caixia Li a, Bing Qi b, Anquan Ji a, Xiulan Xu a, Lan Hu a,* a
Institute of Forensic Science, Ministry of Public Security, Beijing 100038, China b Chinese Peoples Public Security University, Beijing 100038, China Received 4 August 2009; accepted 7 August 2009
Abstract Micromanipulation method was combined with the AmpliGrid LV-PCR system to select and detect single cells, in order to provide a possible solution for biological mixtures and trace samples. Three fresh buccal cells could be completely genotyped by two STR kits. Sixty parallel single cell LV-PCRs were performed using Identifiler1, 13 complete profiles (21.7%) and 13 acceptable profiles (13–15 loci) were obtained. Seventy single cells were typed by MiniFiler1, showing 48.6% full profiles and 18.6% acceptable profiles (6–8 loci). Two mock casework samples, cup and chewing gum, were assayed by single cell LV-PCR using MiniFiler1. Three out of four repeat experiments for the cup sample were fully genotyped. Twice of four chewing gum experiments yielded complete profiles. We re-analyzed one casework sample which had been shown to be a mixed profile tested by routine method, while single-person profile was obtained by the new method. These results showed great promise for mixtures and trace DNA analysis. Successful capture of intact cell is the key to the experiment. Replicate experiments are also necessitated to get reliable profiles. # 2009 Elsevier Ireland Ltd. All rights reserved. Keywords: Mixture sample; Buccal cell; Micromanipulation; LV-PCR
1. Introduction DNA genotyping plays an important role in forensic science nowadays, but trace samples and biological mixtures are problematic and quite challenging for successful detection. In order to solve the problems, micromanipulation and laser microdissection (LCM) have been introduced and greatly improved the capability to select single cell for detection [1,2]. In recent years, we have studied both methods for forensic sample detection [3–5]. To enhance the sensitivity of single cell PCR reactions, we combined micromanipulation method with an on chip-low volume PCR system (LV-PCR), which was originally developed for single cell analysis [6]. 2. Materials and methods Fresh buccal swabs, mock caseworks (cup and chewing gum) were collected from different volunteers. One cigarette butt from a rape case was provided by our laboratory. Cotton swabs were used to collect cells from the cup ridge and the * Corresponding author. Tel.: +86 10 66269503; fax: +86 10 66269503. E-mail addresses: [email protected] (C. Li), [email protected] (L. Hu). 1875-1768/$ – see front matter # 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2009.08.016
cigarette butt. Samples were suspended in TNE solution, and the suspensions were smeared on microscope slides. Micromanipulation was operated under inverted microscope (Olympus) with Transfer Man NK2 micromanipulator (Eppendorf). Low volume-PCR (LV-PCR) was performed with AG480F AmpliGrid slide on AmpliSpeed Cycler (Advalytix). Buccal cells were captured and transferred to AmpliGrid reaction sites. After incubation with 0.75 ml of proteinase K (0.4 mg/ml) for 40 min at 56 8C and 10 min at 99 8C, 0.75 ml of PCR master mix of Identifiler1 or (and) MiniFiler1 (Applied Biosystem) was added. PCR conditions were as follows: 95 8C for 11 min; 28 cycles of 94 8C for 20 s, 59 8C for 1.25 min, 72 8C for 1.25 min; followed by 60 8C for 45 min. Negative controls (no template) were performed on each AmpliGrid. PCR products were transferred to 10 ml of formamide and applied to a 3130 XL Genetic Analyzer (Applied Biosystem). 3. Results 3.1. Single cell detection Three fresh buccal cells could be completely genotyped by both kits. Sixty parallel single cell LV-PCRs were performed using Identifiler1, 13 complete allelic profiles (21.7%) and 13
C. Li et al. / Forensic Science International: Genetics Supplement Series 2 (2009) 516–517 Table 1 Single cell LV-PCR result amplified by Identifiler1 and MiniFiler1 kit. No. of genotyped loci
Identifiler1 16
MiniFiler1
13–15 9–12 0–8 Sum (n) 9
No. of test 13 13 No. of test with 2 5 artificial alleles Call rate (%) 21.7 21.7
15 5
19 4
25
31.7
60 16
34 5
Table 2 The result of the cigarette butt amplified by Identifiler1 kit. Locus
6–8 3–5 0–2 Sum (n) 13 10
8 6
15 3
70 24
48.6 18.6 11.4 21.4
acceptable profiles (13–15 loci) were obtained. Seventy single cells were typed by MiniFiler1, showing 48.6% full profiles and 18.6% acceptable profiles (6–8 loci). The results are listed in Table 1. 3.2. Mock casework sample study Two mock casework samples, cup and chewing gum, were assayed by single cell LV-PCR using MiniFiler1. Three out of four repeat experiments for the cup sample were successfully typed. Allele dropout of D13S317 was observed in one test. Twice of four chewing gum experiments gave complete profiles. Allele dropout of D7S820 and D18S51 was observed in one test. One additional allele of D16S539 was found in another test. Based on Gill’s guidelines [7] where alleles should be confirmed in a minimum of two independent PCR reactions we pooled the results from these independent assays to obtain a consecutive genotype. The genotype is proved to be accurate. 3.3. Casework sample analysis One cigarette butt was re-analyzed by our new method. The sample was tested to be a mixture profile by routine method. Three cells were captured and detected by Identifiler1, and six replicates were conducted. We pooled the six results, and a single-person profile was obtained (Table 2), which was in concordance with the profile obtained from one of the condom of the criminal scene. All the alleles could be found in the previous mixture profile.
517
D8S1179 D21S11 D7S820 CSF1PO D3S1358 TH01 D13S317 D16S539 D2S1338 D19S433 vWA TPOX D18S51 AMEL D5S818 FGA
No. of test 1
2
3
4
5
6
10, 14 29, 32.2 10, 12 10, 11 16 9 8, 11 9, 14 20 14, 15.2 18, 20 8 12, 16 X, Y 11 23, 24
10, 14 29, 32.2 10,12 11 16 9 11 9 – 14, 15.2 20 8 16 X, Y 11 23
14 29, 32.2 10, 12 11 16 9 8 – 20 14, 15.2 18, 20 8 16 X, Y 11 23
9, 10, 14 29 10 – 16 9 11 9, 14 19, 20 14, 15 18, 20 8 12, 16 X, Y 11 23, 24
10, 14 29, 32.2 12 10 16 – 8, 11 14 19 14, 15.2 20 8 – X, Y 11,12 23
10, 14 29, 32.2 12 – 16 9 8, 11 9, 14 – 14, 15.2 18, 20 8 16 X, Y 11 23, 24
Pooling result 10, 14 29, 32.2 10, 12 10, 11 16 9 8, 11 9, 14 19, 20 14, 15.2 18, 20 8 12, 16 X, Y 11 23, 24
the micromanipulation and LV-PCR experiment to avoid contamination and ensure the accuracy of the result. Further study on casework samples detection is ongoing now. Conflict of interest Authors disclose no conflicts of interest. Funding source This study was supported by the grant of Key Projects in the National Science & Technology Pillar Program in the Eleventh Five-year Plan Period (no. 2006BAK07B01). Acknowledgements We would like to thank individuals within Chinese Peoples Public Security University who contribute buccal swabs and mock casework samples for analysis. We thank Dr. Wolfgang Mann for proofreading of the manuscript.
4. Discussion
References
The study demonstrates that the micromanipulation-LV-PCR combination is highly sensitive for single cell assay. Even a single buccal cell can be genotyped completely sometimes (21.7%), which showed great promise for forensic mixtures and trace sample analysis. For the cigarette butt, we obtained a singleperson profile out of the mixed sample, which is a further step towards resolving the biological mixtures problem. We failed to obtain the profile of another person, because the sample had been assayed six months ago, meaning the sample had also been collected then. Only a few cells were left and the DNA degradation of course influences the final result. In fact, successful capture of intact cell is the key to the experiment. Casework samples are difficult to be detected compared with fresh buccal cells, because of the DNA degradation and the influence of different carrier materials, thereby two or three cells need to be captured, and replicate experiment is necessitated to get a reliable profile. In addition, we have established a BSL-2 bio-safety laboratory for
[1] A.T. Findlay, P. Quirke, R. Frazier, A. Urquhart, DNA fingerprinting from single cells, Nature 389 (1997) 555–556. [2] K. Elliott, D.S. Hill, C. Lambert, T.R. Burroughes, P. Gill, Use of laser microdissection greatly improves the recovery of DNA from sperm on microscope slides, Forensic Sci. Int. 137 (2003) 28–36. [3] L. Hu, An.Q. Ji, C.X. Li, Q. Su, X. Li, Micromanipulation separation of cells for short tandem repeats analysis, in: 18th IAFS 246, 2008, p. 85 (Abstract). [4] X. Li, L. Hu, H.L. Guo, Zh.F. Liu, Zh. Tu, R.H. Mi, The exploring of a method for isolating sperm cells from mixture sample, Chin. J. Forensic Med. 5 (2006) 263–265. [5] B. Qi, W.Sh. Li, B. Liu, L.J. Zhang, H.D. Liu, J.L. Yi, C.X. LI, L. Hu, A discussion of laser capture microdissection in forensic application, Evidence Sci. 16 (2008) 764–768. [6] C. Proff, M.A. Rothschild, P.M. Schneider, Low volume PCR (LV-PCR) for STR typing of forensic casework samples, in: A. Amorium, F. Corte-real, N. Morling (Eds.), Progress in Forensic Genetics, Elsevier, Amsterdam, 2006, pp. 645–647. [7] P. Gill, J. Whitaker, C. Flaxman, N. Brown, J. Buckleton, An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA, Forensic Sci. Int. 112 (2000) 17–40.
Forensic Science International: Genetics Supplement Series 2 (2009) 516–517 www.elsevier.com/locate/FSIGSS
Research article
The combination of single cell micromanipulation with LV-PCR system and its application in forensic science Caixia Li a, Bing Qi b, Anquan Ji a, Xiulan Xu a, Lan Hu a,* a
Institute of Forensic Science, Ministry of Public Security, Beijing 100038, China b Chinese Peoples Public Security University, Beijing 100038, China Received 4 August 2009; accepted 7 August 2009
Abstract Micromanipulation method was combined with the AmpliGrid LV-PCR system to select and detect single cells, in order to provide a possible solution for biological mixtures and trace samples. Three fresh buccal cells could be completely genotyped by two STR kits. Sixty parallel single cell LV-PCRs were performed using Identifiler1, 13 complete profiles (21.7%) and 13 acceptable profiles (13–15 loci) were obtained. Seventy single cells were typed by MiniFiler1, showing 48.6% full profiles and 18.6% acceptable profiles (6–8 loci). Two mock casework samples, cup and chewing gum, were assayed by single cell LV-PCR using MiniFiler1. Three out of four repeat experiments for the cup sample were fully genotyped. Twice of four chewing gum experiments yielded complete profiles. We re-analyzed one casework sample which had been shown to be a mixed profile tested by routine method, while single-person profile was obtained by the new method. These results showed great promise for mixtures and trace DNA analysis. Successful capture of intact cell is the key to the experiment. Replicate experiments are also necessitated to get reliable profiles. # 2009 Elsevier Ireland Ltd. All rights reserved. Keywords: Mixture sample; Buccal cell; Micromanipulation; LV-PCR
1. Introduction DNA genotyping plays an important role in forensic science nowadays, but trace samples and biological mixtures are problematic and quite challenging for successful detection. In order to solve the problems, micromanipulation and laser microdissection (LCM) have been introduced and greatly improved the capability to select single cell for detection [1,2]. In recent years, we have studied both methods for forensic sample detection [3–5]. To enhance the sensitivity of single cell PCR reactions, we combined micromanipulation method with an on chip-low volume PCR system (LV-PCR), which was originally developed for single cell analysis [6]. 2. Materials and methods Fresh buccal swabs, mock caseworks (cup and chewing gum) were collected from different volunteers. One cigarette butt from a rape case was provided by our laboratory. Cotton swabs were used to collect cells from the cup ridge and the * Corresponding author. Tel.: +86 10 66269503; fax: +86 10 66269503. E-mail addresses: [email protected] (C. Li), [email protected] (L. Hu). 1875-1768/$ – see front matter # 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2009.08.016
cigarette butt. Samples were suspended in TNE solution, and the suspensions were smeared on microscope slides. Micromanipulation was operated under inverted microscope (Olympus) with Transfer Man NK2 micromanipulator (Eppendorf). Low volume-PCR (LV-PCR) was performed with AG480F AmpliGrid slide on AmpliSpeed Cycler (Advalytix). Buccal cells were captured and transferred to AmpliGrid reaction sites. After incubation with 0.75 ml of proteinase K (0.4 mg/ml) for 40 min at 56 8C and 10 min at 99 8C, 0.75 ml of PCR master mix of Identifiler1 or (and) MiniFiler1 (Applied Biosystem) was added. PCR conditions were as follows: 95 8C for 11 min; 28 cycles of 94 8C for 20 s, 59 8C for 1.25 min, 72 8C for 1.25 min; followed by 60 8C for 45 min. Negative controls (no template) were performed on each AmpliGrid. PCR products were transferred to 10 ml of formamide and applied to a 3130 XL Genetic Analyzer (Applied Biosystem). 3. Results 3.1. Single cell detection Three fresh buccal cells could be completely genotyped by both kits. Sixty parallel single cell LV-PCRs were performed using Identifiler1, 13 complete allelic profiles (21.7%) and 13
C. Li et al. / Forensic Science International: Genetics Supplement Series 2 (2009) 516–517 Table 1 Single cell LV-PCR result amplified by Identifiler1 and MiniFiler1 kit. No. of genotyped loci
Identifiler1 16
MiniFiler1
13–15 9–12 0–8 Sum (n) 9
No. of test 13 13 No. of test with 2 5 artificial alleles Call rate (%) 21.7 21.7
15 5
19 4
25
31.7
60 16
34 5
Table 2 The result of the cigarette butt amplified by Identifiler1 kit. Locus
6–8 3–5 0–2 Sum (n) 13 10
8 6
15 3
70 24
48.6 18.6 11.4 21.4
acceptable profiles (13–15 loci) were obtained. Seventy single cells were typed by MiniFiler1, showing 48.6% full profiles and 18.6% acceptable profiles (6–8 loci). The results are listed in Table 1. 3.2. Mock casework sample study Two mock casework samples, cup and chewing gum, were assayed by single cell LV-PCR using MiniFiler1. Three out of four repeat experiments for the cup sample were successfully typed. Allele dropout of D13S317 was observed in one test. Twice of four chewing gum experiments gave complete profiles. Allele dropout of D7S820 and D18S51 was observed in one test. One additional allele of D16S539 was found in another test. Based on Gill’s guidelines [7] where alleles should be confirmed in a minimum of two independent PCR reactions we pooled the results from these independent assays to obtain a consecutive genotype. The genotype is proved to be accurate. 3.3. Casework sample analysis One cigarette butt was re-analyzed by our new method. The sample was tested to be a mixture profile by routine method. Three cells were captured and detected by Identifiler1, and six replicates were conducted. We pooled the six results, and a single-person profile was obtained (Table 2), which was in concordance with the profile obtained from one of the condom of the criminal scene. All the alleles could be found in the previous mixture profile.
517
D8S1179 D21S11 D7S820 CSF1PO D3S1358 TH01 D13S317 D16S539 D2S1338 D19S433 vWA TPOX D18S51 AMEL D5S818 FGA
No. of test 1
2
3
4
5
6
10, 14 29, 32.2 10, 12 10, 11 16 9 8, 11 9, 14 20 14, 15.2 18, 20 8 12, 16 X, Y 11 23, 24
10, 14 29, 32.2 10,12 11 16 9 11 9 – 14, 15.2 20 8 16 X, Y 11 23
14 29, 32.2 10, 12 11 16 9 8 – 20 14, 15.2 18, 20 8 16 X, Y 11 23
9, 10, 14 29 10 – 16 9 11 9, 14 19, 20 14, 15 18, 20 8 12, 16 X, Y 11 23, 24
10, 14 29, 32.2 12 10 16 – 8, 11 14 19 14, 15.2 20 8 – X, Y 11,12 23
10, 14 29, 32.2 12 – 16 9 8, 11 9, 14 – 14, 15.2 18, 20 8 16 X, Y 11 23, 24
Pooling result 10, 14 29, 32.2 10, 12 10, 11 16 9 8, 11 9, 14 19, 20 14, 15.2 18, 20 8 12, 16 X, Y 11 23, 24
the micromanipulation and LV-PCR experiment to avoid contamination and ensure the accuracy of the result. Further study on casework samples detection is ongoing now. Conflict of interest Authors disclose no conflicts of interest. Funding source This study was supported by the grant of Key Projects in the National Science & Technology Pillar Program in the Eleventh Five-year Plan Period (no. 2006BAK07B01). Acknowledgements We would like to thank individuals within Chinese Peoples Public Security University who contribute buccal swabs and mock casework samples for analysis. We thank Dr. Wolfgang Mann for proofreading of the manuscript.
4. Discussion
References
The study demonstrates that the micromanipulation-LV-PCR combination is highly sensitive for single cell assay. Even a single buccal cell can be genotyped completely sometimes (21.7%), which showed great promise for forensic mixtures and trace sample analysis. For the cigarette butt, we obtained a singleperson profile out of the mixed sample, which is a further step towards resolving the biological mixtures problem. We failed to obtain the profile of another person, because the sample had been assayed six months ago, meaning the sample had also been collected then. Only a few cells were left and the DNA degradation of course influences the final result. In fact, successful capture of intact cell is the key to the experiment. Casework samples are difficult to be detected compared with fresh buccal cells, because of the DNA degradation and the influence of different carrier materials, thereby two or three cells need to be captured, and replicate experiment is necessitated to get a reliable profile. In addition, we have established a BSL-2 bio-safety laboratory for
[1] A.T. Findlay, P. Quirke, R. Frazier, A. Urquhart, DNA fingerprinting from single cells, Nature 389 (1997) 555–556. [2] K. Elliott, D.S. Hill, C. Lambert, T.R. Burroughes, P. Gill, Use of laser microdissection greatly improves the recovery of DNA from sperm on microscope slides, Forensic Sci. Int. 137 (2003) 28–36. [3] L. Hu, An.Q. Ji, C.X. Li, Q. Su, X. Li, Micromanipulation separation of cells for short tandem repeats analysis, in: 18th IAFS 246, 2008, p. 85 (Abstract). [4] X. Li, L. Hu, H.L. Guo, Zh.F. Liu, Zh. Tu, R.H. Mi, The exploring of a method for isolating sperm cells from mixture sample, Chin. J. Forensic Med. 5 (2006) 263–265. [5] B. Qi, W.Sh. Li, B. Liu, L.J. Zhang, H.D. Liu, J.L. Yi, C.X. LI, L. Hu, A discussion of laser capture microdissection in forensic application, Evidence Sci. 16 (2008) 764–768. [6] C. Proff, M.A. Rothschild, P.M. Schneider, Low volume PCR (LV-PCR) for STR typing of forensic casework samples, in: A. Amorium, F. Corte-real, N. Morling (Eds.), Progress in Forensic Genetics, Elsevier, Amsterdam, 2006, pp. 645–647. [7] P. Gill, J. Whitaker, C. Flaxman, N. Brown, J. Buckleton, An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA, Forensic Sci. Int. 112 (2000) 17–40.