Validation of sensitive human leukocyte antigen-sequence-specific primer and probe typing in forensic DNA examination

Legal Medicine 8 (2006) 203–209 www.elsevier.com/locate/legalmed

Validation of sensitive human leukocyte antigen-sequence-specific primer and probe typing in forensic DNA examination Masao Ota a,*, Kazunori Shimada b, Hideki Asamura a, Kayoko Takayanagi a, Yoshihiko Katsuyama c, Hirofumi Fukushima a a

Department of Legal Medicine, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan b Genome Science Laboratories, Co., LTD, Fukushima, Japan c Department of Pharmacy, Shinshu University Hospital, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan Received 21 December 2005; received in revised form 7 March 2006; accepted 8 March 2006 Available online 27 June 2006

Abstract Validation studies were carried out with the commercially available HLA typing kit using a PCR-SPP (sequence-specific primer and probe) technique. This technique has made it possible to type class I (HLA-A and -B) and class II (HLA-DRB1 and -DQB1) alleles at low-resolution level with total 10 ng of template DNA, in addition to amplify directly from various forms of blood samples without DNA isolation procedure. Experimental examinations with bloodstains smeared on cotton cloth that were a week to 3 months old, bloodstains on gauze stored for 18 years, and buccal cells revealed that this HLA-SPP typing kit is a sensitive and reliable method for forensic investigations. q 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: HLA typing; PCR-SPP technique; Direct PCR; Validation study; Forensic samples

1. Introduction The advent of recent molecular technology using highly polymorphic markers such as microsattellites and minisattellites has brought a remarkable progress on forensic identity testing and parentage determination [1–4]. For the majority of caseworks involving DNA profiling, most of forensic laboratories now routinely use the commercially developed multiplex short tandem repeat (STR) amplification kits together with automated fluorescence-based allele detection methodology [5–7]. This is mainly caused due to the inherent the advantages of this reliable method and as well as the great power for individualization as the probability of a random match between two unrelated people (discrimination power and polymorphic information content). Human leukocyte antigen (HLA) alleles also

* Corresponding author. Tel.: C81 263 37 3217; fax: C81 263 37 3084. E-mail address: [email protected] (M. Ota).

1344-6223/$ - see front matter q 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.legalmed.2006.03.001

display an extreme degree of genetic polymorphisms [8,9] (372 alleles in HLA-A, 190 alleles in HLA-B and 490 alleles in HLA-DRB). These highly polymorphic loci are equally useful as human genetic markers in forensic investigations [10]. However, it has been difficult to simultaneously type class I (HLA-A and -B) and class II (HLA-DRB1) alleles from an extremely small amount of DNA, such as that from a single hair, a piece of nail, formalin-fixed and paraffin-embedded tissues, and the like forensic specimens. Recently, a commercially available HLA typing kit (Genome Science Laboratories, Co, Fukushima, Japan) using a PCR-SPP (sequence-specific primer and probe) technique has made it possible to type HLA class-I (-A and -B) and class-II (-DRB1) alleles at low-resolution level from the minimum amount of DNA (of 10 ng). In the present study, we have investigated the applicability of this typing kit to forensic practice using DNA samples extracted form stains of 1 ml of blood on cotton cloth stored for a week to 3 months, bloodstains stored at room temperature for 18 years and buccal cells.

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2. Materials and methods 2.1. Samples One microliter of whole blood was used for HLA-SPP typing as a reference. One microliter of blood samples from three different individuals was smeared on cotton cloth, and thus-prepared bloodstains were typed a week, 1–3 months later. HLA types of these samples were also decided by different typing kits (Micro SSP JPN DNA typing Tray, One Lambda, CA, USA). HLA types of the samples were as follows, no. 1: A24A26/B13B61/DR9DR14, no. 2: A24A26/ B7B61/DR1DR12 and no. 3: A2A33/B61B44/DR13DR14. As actual forensic identification cases, four kinds of aged bloodstains stored at room temperature for 18 years were subjected to the HLA-DNA typing. Serologically defined HLA types of the bloodstains were A24A33/ B51B40/DR6DR9 for the bloodstain no. 1, A24A31/ B7B35 of no. 2, A2A31/B46B51/DR4DR8 for no. 3 and A2A24/B46B55/DR2DR8 for no. 4. DR type of the bloodstain no. 2 could not be determined.

the suspension was mixed directly with 40.3 ml of PCR mixture containing 20 ml of direct amplification buffer [11,12]. DNA extraction and first PCR procedure from bloodstains smeared on cotton cloth and gauze was followed by a newly developed procedure (Fig. 1). Twenty microliter of 0.1% Tween solvent was poured in each HLA locus specific tube. Then, a 1!3 mm squared fragment or a punched fragment with 1.0 mm in diameter of bloodstains on cotton cloth, or three pieces of thread raveled out from the bloodstains on gauze by tweezers was incubated in a Tween solvent more than 5 min at room temperature. Cytobrush samples collected from buccal cells by CytoSofte Brush (MPC, CA, USA) were suspended in 300 ml of 0.1% Tween solution, 20 ml of homogenous suspension were moved to the reaction tube. After addition of 20 ml of the first master mix containing locus specific primers (supplied with the HLA typing kit), 0.3 ml (1.5 units) of Taq polymerase, and 20 ml of direct amplification buffer (supplied with the kit) into each tube, the first PCR was performed as described in Section 2.3. 2.3. HLA-SSP typing

2.2. Sample preparation DNA was isolated from various samples with a reagent cocktail supplied with the kit. One microliter of whole blood was suspended in 100 ml of distilled water, and 20 ml of

This unique HLA typing is consisted of dual PCR amplification and hybridization of PCR products with allele specific probes binding to the wall of microplate wells. The first PCR is performed with locus specific generic primers:

Fig. 1. HLA-A and -DRB1 allele typing are exampled in the process of HLA-SPP method. Locus specific generic primers are used in the first PCR. Allele specific primers are employed in the second PCR. Sixteen allele specific probes in HLA-A and -DRB1 typing are anchored on the wall of microplate. TMB, 3,3 0 ,5,5 0 -tetramethylbenzidine; HRP, horseradish peroxidase; SA, streptavidin.

M. Ota et al. / Legal Medicine 8 (2006) 203–209

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Table 1 A time course study on HLA-SPP method derived from bloodstains on the cotton cloth A

A1 A2 A3 A11 A24 A25 A26 A2603 A29 A30 A31 A32 A33 A43 A68 A74

1

2

3

1 Week 24,26

1 Month

2 Month

3 Month

1 Week 24,2603

1 Month

2 Month

3 Month

1 Week 2,33

1 Month

2 Month

3 Month

0.035 0.034 0.048 0.041 (C) 0.032 (C) 0.139 0.027 0.035 0.037 0.041 0.031 0.045 0.045 0.077

0.042 0.036 0.044 0.040 (C) 0.031 (C) 0.180 0.034 0.043 0.031 0.270 0.031 0.037 0.046 0.143

0.030 0.030 0.027 0.029 (C) 0.026 (C) 0.106 0.032 0.051 0.033 0.026 0.029 0.028 0.058 0.070

0.042 0.040 0.072 0.056 3.559 0.049 3.536 0.107 0.039 0.064 0.045 0.163 0.039 0.038 0.055 0.084

0.035 0.030 0.040 0.039 (C) 0.027 0.029 1.650 0.027 0.045 0.034 0.106 0.028 0.037 0.045 0.109

0.082 0.041 0.038 0.036 (C) 0.028 0.043 2.059 0.027 0.081 0.029 0.042 0.028 0.042 0.069 0.140

0.033 0.029 0.027 0.032 (C) 0.024 0.036 1.300 0.031 0.046 0.029 0.025 0.028 0.032 0.071 0.088

0.045 0.039 0.052 0.062 3.530 0.036 0.044 1.517 0.034 0.043 0.042 0.045 0.036 0.047 0.054 0.080

0.033 (C) 0.042 0.041 0.033 0.030 0.034 0.098 0.033 0.222 0.036 0.045 2.077 0.052 0.102 0.077

0.030 (C) 0.038 0.035 0.028 0.025 0.030 0.137 0.026 0.045 0.030 0.041 2.803 0.032 0.165 0.125

0.034 (C) 0.034 0.034 0.029 0.026 0.027 0.048 0.028 0.031 0.027 0.039 1.299 0.114 0.057 0.048

0.045 3.557 0.050 0.051 0.037 0.033 0.038 0.086 0.034 0.041 0.044 0.043 2.539 0.036 0.083 0.081

0.094 3.934 0.027 0.062 0.026 0.029 0.029 0.022 0.039 0.038 0.028 0.026 0.028 0.218 0.028 3.445 0.031 0.026 0.050 0.034 0.030 0.023 0.028 0.036 0.028 0.066 0.275 0.037 0.028 0.188 0.028 0.024

0.125 (C) 0.031 0.034 0.027 0.030 0.029 0.023 0.061 0.030 0.314 0.028 0.037 0.352 0.087 3.557 0.028 0.031 0.030 0.033 0.036 0.027 0.029 0.051 0.028 0.072 0.428 0.032 0.025 0.544 0.026 0.026

2.049 0.042 0.027 1.369 3.672 0.350 0.038 0.025 0.315 0.042 0.031 0.049 0.038 0.587 0.026 0.025 0.035 0.031 0.030 0.033 0.038 0.032 0.031 0.051 0.028 0.050 0.552 0.042 0.029 0.468 0.034 0.025

1.764 0.037 0.026 0.762 3.842 0.089 0.034 0.026 0.058 0.038 0.029 0.033 0.026 0.183 0.088 0.028 0.027 0.027 0.029 0.033 0.026 0.026 0.027 0.042 0.043 0.035 0.472 0.032 0.026 0.354 0.031 0.025

1.523 0.158 0.033 0.583 0.585 0.085 0.045 0.043 0.061 0.175 0.036 0.033 0.040 0.058 0.042 0.052 0.033 0.035 0.034 0.042 0.036 0.046 0.042 0.057 0.030 0.046 0.307 0.036 0.041 0.166 0.036 0.040

0.141 0.053 0.035 0.048 0.035 0.033 0.035 0.030 0.072 0.076 0.070 0.034 0.036 0.110 0.042 3.556 1.439 0.037 0.036 0.048 0.036 0.033 0.032 0.040 0.030 0.044 0.224 0.044 0.032 0.041 0.041 0.032

0.083 0.042 0.028 0.030 0.030 0.031 0.030 0.024 0.048 0.030 0.029 0.025 0.025 0.142 0.028 3.723 0.931 0.028 0.029 0.032 0.030 0.029 0.031 0.045 0.034 0.050 0.157 0.032 0.025 0.106 0.036 0.032

0.090 0.118 0.033 0.040 0.032 0.043 0.036 0.046 0.051 0.060 0.075 0.039 0.074 0.051 0.041 3.472 0.653 0.040 0.045 0.041 0.052 0.036 0.033 0.050 0.032 0.059 0.314 0.041 0.037 0.050 0.041 0.036

0.061 0.090 0.027 0.050 0.129 0.025

0.044 0.057 0.031 0.033 0.079 0.028 (continued on

B

13,61

B7 B13 B14 B62 B15(62.75) B75 B15(70) B15(63) B18 B27 B35 B37 B38 B39 B40(60) B40(61) B44 B46 B47 B48 B51 B52 B53 B54 B55 B55.56 B56 B58 B59 B67 B78 B81

0.076 (C) 0.027 0.036 0.032 0.028 0.032 0.029 0.056 0.030 0.330 0.039 0.032 0.136 0.039 3.658 0.024 0.027 0.032 0.048 0.031 0.026 0.044 0.043 0.022 0.040 0.247 0.042 0.030 0.026 0.031 0.028

DR

9,14-1

DR1 DR3 DR4 DR7 DR8 DR9

0.029 0.027 0.028 0.034 0.026 3.709

7,62 0.080 3.610 0.044 0.060 0.072 0.045 0.045 0.047 0.050 0.052 0.041 0.041 0.052 0.041 0.064 3.213 0.041 0.033 0.040 0.049 0.043 0.033 0.047 0.072 0.033 0.035 0.333 0.031 0.043 0.041 0.039 0.041

1.505 0.041 0.024 1.390 3.892 0.134 0.038 0.023 0.071 0.028 0.029 0.041 0.032 0.327 0.030 0.027 0.029 0.031 0.028 0.032 0.029 0.031 0.039 0.046 0.021 0.041 0.500 0.035 0.035 0.337 0.032 0.027

44,61

1,12 0.027 0.030 0.029 0.052 0.205 3.591

0.031 0.031 0.039 0.041 0.048 2.703

0.039 0.042 0.034 0.033 0.048 3.571

(C) 0.028 0.083 0.144 0.038 0.042

0.082 0.046 0.023 0.048 0.025 0.035 0.042 0.025 0.078 0.029 0.379 0.040 0.050 0.282 0.038 3.688 1.387 0.025 0.029 0.041 0.059 0.036 0.039 0.042 0.030 0.059 0.305 0.037 0.070 0.086 0.049 0.045 13-1, 14-3

(C) 0.028 0.022 0.025 0.056 0.027

(C) 0.037 0.032 0.034 0.034 0.044

(C) 0.032 0.031 0.034 0.031 0.035

0.060 0.039 0.029 0.054 0.027 0.038

0.030 0.068 0.037 0.038 0.035 0.031 next page)

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Table 1 (continued) DR

9,14-1

DR10 DR11 DR12 DR13-1 DR13-2 DR14-1 DR14-2 DR14-3 DR15 DR16

0.030 0.028 0.030 0.027 0.028 3.604 0.029 0.028 0.033 0.027

1,12 0.031 0.036 0.027 0.033 0.029 3.575 0.086 0.025 0.028 0.037

0.030 0.028 0.036 0.046 0.035 2.974 0.036 0.033 0.037 0.031

0.030 0.033 0.041 0.153 0.033 3.074 0.036 0.034 0.034 0.030

0.029 0.028 1.965 0.027 0.027 0.028 0.032 0.028 0.031 0.025

13-1, 14-3 0.028 0.030 1.573 0.033 0.027 0.027 0.034 0.053 0.039 0.027

0.031 0.030 2.258 0.043 0.031 0.030 0.033 0.029 0.032 0.029

0.032 0.031 1.651 0.042 0.033 0.031 0.033 0.032 0.034 0.030

0.028 0.026 0.121 2.961 0.176 0.047 0.145 (C) 0.030 0.028

0.025 0.029 0.033 3.260 0.077 0.034 0.299 (C) 0.025 0.031

0.028 0.046 0.226 2.210 0.073 0.042 0.121 3.894 0.037 0.027

0.032 0.033 0.030 3.197 0.075 0.037 0.129 3.699 0.034 0.031

(C), Means strong positive reaction (O4.000), bold font shows a positive reaction.

exon 2 of HLA-A locus, exons 2 and 3 of HLA-B locus and exon 2 of HLA-DRB1 locus are amplified. The second PCR is carried out using allele specific primer pairs (16 primer pairs each for HLA-A and -DRB1, and 32 primer pairs for HLA-B). Either of allele specific primer pair is labeled with biotin. After the alkaline treatment of PCR products, the denatured single strand DNA is hybridized to allele specific probes, which are immobilized to the wall of microplate wells. Through biotin–streptavidin linked enzyme reaction, the visible color is measured by a microplate reader at 450 nm wavelength. Generally, the cut off value is decided less than 0.5. 2.4. PCR reaction PCR reaction was carried out according to the manufacturer’s instructions. The first PCR amplification was carried out in a total 60.3 ml of reaction mixture, with 20 cycles of denaturing at 95 8C for 30 s, annealing at 58 8C for 30 s and extension at 72 8C for 30 s. After PCR, 40 ml of distilled water and 1.5 ml (5 units/ml) of Taq polymerase were added to the first PCR mixture and mixed well with a vortex. Then 5 ml of this mixture was moved to the microplate well and mixed with 10 ml of the second master mixture containing biotin labeled allele specific primers and dNTPs (supplied with the HLA typing kit). The second PCR amplification was performed in a total 15 ml of reaction mixture, with initial incubation at 50 8C for 5 min and denaturation at 95 8C for 1 min, followed by 50 cycles of denaturation at 95 8C for 30 s, annealing at 65 8C for 30 s and extension at 72 8C for 30 s.

3. Results and discussion HLA types by PCR-SPP using 1 ml of fresh blood samples from randomly selected healthy individuals were completely matched with serologically defined results obtained by the lymphocyte cytotoxicity test (LCT). Manufacturer’s protocol recommends the most suitable DNA quantity for exact typing is 2 ng in the first PCR mixture, whereas excess DNA may lead to non-specific results. All our experiments using fresh bloodstains to evaluate the reliability and detection power in the HLA-SPP

typing kit were undertaken using 1 ml of blood on cotton cloth. DNA typing result obtained from 1 ml of three different bloodstains was completely in accordance with serologically defined types by LCT (Table 1). HLA typing was possible at 1 week, 1–3 month intervals after preparation of bloodstains in all samples. All positive reactions showed more than 0.5 values in absorbance at 450 nm. Although HLA types are actually decided by DNA based method, their descriptions are pursuant to serologically based typing. HLA-DR13-1, DR13-2, DR14-1, DR14-2, and DR14-3 in Table 1 indicate the amplification of group specific DRB1* alleles as follows: DR13-1 for DRB1*1301, 1302, 1305 and 1306 alleles, DR13-2 for DRB1*1303 and 1304 alleles, DR14-1 for DRB1*1401, 1407 and 1408 alleles, DR14-2 for DRB1*1402, 1403 and 1406 alleles, DR14-3 for DRB1* 1405 allele. In HLA-B typing of sample, no. 2, the B56 exhibited relatively large value in absorbance compared with other negative wells. This phenomenon resulted from close reactivity with some amplification primers or group specific probes, which are described in manufacturer’s cautionary statement. The real values in absorbance and HLA types from 18 aged bloodstains on gauze cloth are shown in Table 2. Bloodstain no. 1 was typed A24A33/B51B60(40)/DR9D131, bloodstain no. 2 was A24A31/B7B35/DR1DR9, bloodstain no. 3 was A2A31/B46B51/DR4DR8, and bloodstain no. 4 was A2A24/B46B55/DR8DR15. In bloodstains nos. 3 and 4, three strong positive reactions were detected in wells B46, B51 and B62, and B46, B55 and B62, respectively, which might be due to proximity sequences of primers or allele specific probes between B46 and B62. However, B62 positive reactions in both the samples could be determined false by the negative results of B15 (62, 75). The difference in HLA types of bloodstains nos. 1 and 4 between the LCT and SPP methods were caused by the difference in their resolution level. DR6 and DR2 typed by the LCT method are included in DR13-1 and DR15 typed by the lowresolution SPP method, respectively. Buccal cell samples from three different individuals were definitely determined A24A26/B61B62/DR4DR9, A2A/B35B52/DR9DR15, and A24A-/B35B52/DR9DR15 types by the PCR-SPP method.

Table 2 HLA typing results from old aged bloodstains on gauze cloth 2

3

4

1

2

3

4

1

2

3

4

24,33

24,31

2,31

2,24

B

60(40),51

7,35

46,51

46,55

DR

9,13-1

1,9

4,8

8,15

A1 A2 A3 A11 A24 A25 A26 A2603 A29 A30 A31 A32 A33 A43 A68 A74

0.037 0.035 0.043 0.057 (C) 0.027 0.038 0.204 0.033 0.275 0.036 0.043 2.428 0.057 0.097 0.209

0.095 0.029 0.038 0.038 (C) 0.026 0.031 0.187 0.030 0.044 3.719 0.047 0.027 0.097 0.043 0.182

0.030 (C) 0.040 0.040 0.029 0.028 0.030 0.201 0.028 0.089 0.556 0.042 0.028 0.063 0.043 0.223

0.033 (C) 0.040 0.038 (C) 0.026 0.032 0.155 0.028 0.047 0.030 0.039 0.027 0.044 0.053 0.168

B7 B13 B14 B62 B15(62.75) B75 B15(70) B15(63) B18 B27 B35 B37 B38 B39 B40(60) B40(61) B44 B46 B47 B48 B51 B52 B53 B54 B55 B55.56 B56 B58 B59 B67 B78 B81

0.129 0.039 0.039 0.039 0.029 0.033 0.036 0.031 0.122 0.030 0.040 0.035 0.031 0.030 2.135 0.035 0.404 0.025 0.030 0.044 2.980 0.051 0.032 0.058 0.027 0.059 0.400 0.034 0.028 0.047 0.036 0.029

2.621 0.119 0.028 0.030 0.050 0.047 0.049 0.027 0.068 0.030 3.147 0.027 0.029 0.451 0.165 0.031 0.030 0.030 0.027 0.030 0.390 0.025 0.033 0.075 0.029 0.046 0.213 0.031 0.029 0.495 0.032 0.027

0.139 0.039 0.031 0.819 0.031 0.052 0.041 0.031 0.055 0.046 0.030 0.029 0.030 0.027 0.117 0.029 0.030 1.601 0.032 0.033 2.442 0.048 0.037 0.061 0.029 0.032 0.542 0.041 0.028 0.028 0.040 0.032

0.108 0.041 0.031 1.330 0.029 0.042 0.077 0.028 0.069 0.057 0.031 0.055 0.030 0.033 0.061 0.026 0.031 1.674 0.036 0.043 0.030 0.035 0.035 0.047 3.624 0.270 0.200 0.042 0.031 0.061 0.034 0.035

DR1 DR3 DR4 DR7 DR8 DR9 DR10 DR11 DR12 DR13-1 DR13-2 DR14-1 DR14-2 DR14-3 DR15 DR16

0.023 0.025 0.024 0.027 0.030 2.880 0.031 0.034 0.035 2.912 0.091 0.041 0.036 0.027 0.034 0.027

(C) 0.031 0.022 0.029 0.022 2.348 0.025 0.022 0.026 0.029 0.026 0.030 0.026 0.026 0.028 0.116

0.023 0.028 3.935 0.025 3.766 0.024 0.025 0.025 0.029 0.042 0.025 0.030 0.028 0.028 0.030 0.026

0.024 0.026 0.025 0.026 (C) 0.125 0.028 0.027 0.027 0.027 0.026 0.033 0.232 0.029 2.290 0.025

M. Ota et al. / Legal Medicine 8 (2006) 203–209

1 A

(C), Means strong positive reaction (O4.000), bold font shows a positive reaction.

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Table 3 Comparison of statistical parameters between HLA and satellite markers for use in forensic casework Marker HLA

Minisatellites Microsatellites

a

HLA-A HLA-Ba HLA-Cwa HLA-DRBa HLA-DRB1b HLA-DQA1b HLA-DQB1b HLA-DPB1b D1S80 D17S5 ACTBP2 FGA VWA D3S1358 CSF1PO TPOX TH01 D7S820 D13S317 D5S818

HZ

PIC

DP

MEC

Ref.

0.77 0.93 0.78 0.86 0.93 0.75 0.89 0.75 0.91 0.77 0.92 0.86 0.79 0.70 0.73 0.66 0.71 0.76 0.80 0.80

0.72 0.90 0.73 0.82 0.90 0.70 0.85 0.69 0.87 0.83 0.94 0.85 0.76 0.65 0.69 0.60 0.66 0.72 0.77 0.77

0.86 0.94 0.87 0.90 0.94 0.86 0.92 0.86 0.98 0.96 0.99 0.97 0.95 0.86 0.88 0.83 0.86 0.90 0.93 0.93

0.54 0.85 0.50 0.71 0.84 0.52 0.76 0.48 0.77 0.71 0.87 0.73 0.68 0.45 0.50 0.40 0.46 0.54 0.61 0.60

[18]

[19] [20] [21] [22]

All parameters are calculated by Japanese allele frequencies described in references. HZ, expected heterozygosity; PIC, polymorphic information content; MEC, mean exclusion chance. a Low-resolution level. b High-resolution level.

The HLA system exhibits a very high degree of polymorphism with 1794 alleles including 372 HLA-A, 661 HLA-B, 190 HLA-Cw, 481 HLA-DRB1, 28 HLADQA1, and 62 HLA-DQB1 alleles at present [14]. This polymorphism is useful in forensic investigation such as personal identification and parentage testing. The typing system presented here has the power at a level of lowresolution corresponding to serological typing. However, it has high efficiency with high discrimination (in terms of discrimination power and chance of exclusion) in Japanese population (Table 3). HLA typing is generally carried out using fresh blood samples in routine examinations, such as matching for bone marrow or solid organ transplantation, disease-association studies and parentage testing. There are few reports on availability of HLA systems for forensic caseworks using aged, degraded or small amounts of DNA samples [15–17]. The HLA-SPP typing system features extremely high sensitivity and requires no DNA isolation procedure prior to PCR. This might be advantageous to prevent contamination by carryover with labor-intensive steps during DNA extraction. The system therefore promises to provide useful information in forensic caseworks and investigations besides routinely used STR amplification kits. Acknowledgements The authors thank Prof. Siamak Bahram of Center de Recherche d’Immunologie et d’Hematologie, Strasbourg, France, for his comments on drafts of this manuscript. This work was supported in part by research grants from the

Ministry of Education, Culture, Sports, Science and Technology of Japan (Grand-in-Aid for Scientific Research (C), Kakenhi).

References [1] Lygo JE, Johnson PE, Holdaway DJ, Woodroffe S, Whitaker JP, Clayton TM, Kimpton CP, Gill P. The validation of short tandem repeat (STR) loci for use in forensic casework. Int J Leg Med 1994; 107:77–89. [2] Prinz M, Ishii A, Coleman A, Baum HJ, Shaler RC. Validation and casework application of a Y chromosome specific STR multiplex. Forensic Sci Int 2001;120:177–88. [3] Bulter JM, Shen Y, McCord BR. The development of reduced size STR amplicons as tools for analysis of degraded DNA. J Forensci Sci 2003;48:1054–64. [4] Jogling MA, Gill P. Encoded evidence: DNA in forensic analysis. Nat Rev Genet 2004;10:739–51. [5] Cotton EA, Allsop RF, Guest JL, Frazier RRE, Koumi P, Gallow IP, Seager A, Sparke RL. Validation of the AMPF/STRw SGM PlusTM system for use in forensic casework. Forensic Sci Int 2000;112:151–61. [6] Junge A, Lederer T, Braunschweiger G, Madea B. Validation of the multiplex kit genRESMPX-2 for forensic casework analysis. Int J Legal Med 2003;117:317–25. [7] Greenspoon SA, Ban JD, Pablo L, Crouse CA, Kist FG, Tomsey CS, Glessner AL, Mihalacki LR, Long TM, Heidebrecht BJ, Braunstein CA, Greeman DA, Soberalski C, Bruesehoff N, Amin AS, Douglas EK, Shumm JW. Validation and implementation of the PowerPlexw 16BIO system STR multiplex for forensic casework. J Forensic Sci 2004;49:71–80. [8] Robinson J, Waller MJ, Parham P, Bodmer JG, Marsh SGE. IMGT/HLADatabase—a sequence database for the human major histocompatibility complex. Nucleic Acids Res. 2001;29:210–3. [9] Schreuder GM, Hurley CK, Marsh SGE, Lau M, Fermandez-Vina M, Noreen HJ, Setterholm M, Maiers M, The HLA. dictionary 2004:

M. Ota et al. / Legal Medicine 8 (2006) 203–209

[10]

[11]

[12]

[14] [15]

[16]

a summary of HLA-A, -B,-C,-DRB1/3/4/5 and -DQB1 alleles and their association with sereologically defined HLA-A,-B,-C-DR and DQ antigens. Tissue Antigens 2005;65:1–55. Hallenberg C, Morling N. A report of the 1997, 1998 and 1999 paternity testing workshops of the English speaking working group of the international society for forensic genetics. Forensic Sci Int 2001; 116:23–33. Nishimura N, Nakayama T, Tonoike H, Kojima K, Kato S. Direct polymerase chain reaction from whole blood without DNA isolation. Ann Clin Biochem 2000;37:674–80. Nishimura N, Nakayama T, Tonoike H, Kojima K, Shirasaki Y, Kondoh K, Yamada T. Various applications of direct PCR using blood samples. Clin Lab 2002;48:377–84. http://www.ebi.ac.uk/imgt/hla/. Ota M, Katsuyama Y, Liu CY, Arakura A, Fukushima H. Validation of HLA-DR locus typing in forensic specimens by combining PCRSSP with PCR-RFLP. J Forensic Sci 1997;42:929–34. Sato Y, Hayakawa M, Nakajima T, Motani H, Kiuchi M. HLA typing of aortic tissues from unidentified bodies using hot start polymerase chain reaction-sequnce specific primers. Legal Med 2003;5:S191–S3.

209

[17] Keresztury L, Rajczy K, Tauszik T, Gyo´di E, Petra´nyi GG, Falus A. DNA typing revealing high HLA-Cw polymorphism completes availability of major histocompatibility complex loci in forensic medicine. Am J Forensic Med Pathol 2003;24:70–5. [18] Saito S, Ota S, Yamada E, Inoko H, Ota M. Allele frequencies and haplotypic associations defined by allelic DNA typing HLA class I and class II loci in the Japanese population. Tissue Antigens 2000;56:522–9. [19] Sugiyama E, Honda K, Katsuyama Y, Uchiyama S, Tsuchikane A, Ota M, Fukushima H. Allele frequency distribution of the D1S80 (pMCT118) locus polymorphism in the Japanese population by the polymerase chain reaction. Int J Leg Med 1993;106:111–4. [20] Harashima N, Ota M, Katsuyama Y, Sugiyama E, Liu C, Fukushima H. The allele frequency distribution at VNTR locus D17S5(YNZ22) in a Japanese population sample. Jpn J Legal Med 1996;50:237–40. [21] Liu C, Harashima N, Katsuyama Y, Ota M, Arakura A, Fukushima H. ACTBP2 gene frequency distribution and sequencing of the allelic ladder and variants in the Japanese and Chinese populations. Int J Legal Med 1997;110:208–12. [22] Hashiyada M, Itakura Y, Nagashima T, Nata M, Funayama M. Polymorphism of 17 STRS by multiplex analysis in Japanese population. Forensic Sci Int 2003;133:250–3.