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Identification of Severely Burned Bodies From a Vehicle Collision Using the Restriction Fragment Length Polymorphism (RFLP) Technique |
and Lay Hong Seah |
Ministry of Science, Technology, and the Environment Malaysia |
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Ministry of Science, Technology, and the Environment Malaysia |
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Ministry of Science, Technology, and the Environment Malaysia |
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Abstract |
Introduction |
Materials and Methods |
Results and Discussion |
References |
Figures |
The accompanying article describes work conducted by the Department of Chemistry, Malaysia, in its effort to identify 13 severely burned victims of a highway collision involving a tourist bus and a transport truck. Using reference blood samples from their next of kin, all 13 victims were positively identified by the kinship analysis approach using the RFLP (single locus chemiluminescent enhanced probe system) technique.
On December 3, 1996, at approximately 4:45 a.m., a tour bus carrying tourists from Singapore to a mountain resort rammed into the rear of a transport truck that was stopped along a highway approximately 30 km from Kuala Lumpur, Malaysia. Following impact, the bus was engulfed in flames. Thirteen Singaporeans were killed; their bodies were burned beyond recognition. Postmortems on the 13 victims were conducted by pathologists from the Kuala Lumpur Hospital, where a total of 21 blood, bone marrow, and tissue samples were collected and submitted for deoxyribonucleic acid (DNA) analysis. Twenty-four reference blood samples were subsequently collected from their respective family members.
Because the 13 victims were from six different families, and all of the human remains from the accident were referenced to these missing persons, this case presented a closed system for identification. In addition, because the pathologists had confirmed the sex of all the victims, with additional confirmation by the families of the victims and the surviving passengers of the bus, gender discrimination was never an issue.
The DNA Laboratory at the Forensic Division of the Department of Chemistry, Malaysia, was officially operational in November 1994. This was the first case of this nature, complexity, and urgency received by the department. Responding to the need for immediate analysis, a team of six scientific officers positively identified all 13 victims within nine days of receipt of the first batch of samples.
DNA was extracted from each liquid blood, bone marrow, and tissue sample using the phenol/chloroform/isoamyl alcohol extraction after proteinase K digestion (FBI Laboratory 1990; Home Office Forensic Science Services 1990). The DNA was digested with restriction enzyme Hae III, and the digested DNA subsequently electrophoresed in a 0.8 percent agarose gel (Budowle et al. 1990). K562 DNA was used as an internal control marker, and adenovirus DNA digested with Kpn1 restriction enzyme was used as a visual marker during electrophoresis. An analytical sizing marker composed of 30 bands with fragment sizes ranging from 526 to 22,621 base pairs (bp), purchased from Gibco BRL (Rockville, Maryland), was used to determine the size of the alleles of each individual.
DNA analysis using the RFLP (nonisotopic chemiluminescent) technique was performed to detect the Hae III restricted fragment alleles at four genetic lociD2S44, D4S139, D5S110, and D10S28using the probes YNH24 (GenePrint Light), PH30 (Aces), LH1 (Aces), and TBQ7 (GenePrint Light), respectively. GenePrint Light and Aces probes were purchased from Promega (Madison, Wisconsin) and Gibco BRL, respectively.
Table 1 lists the physical identity of the victims and the types of samples submitted by the forensic pathologists performing the autopsies. DNA was extracted from at least one of the samples from each victim for DNA analysis using the RFLP technique.
Victim | Age and Gender | Sample Types |
Body 1 | Child, female, age 10-12 years | Blood, bone marrow |
Body 2 | Adult, male, age 20-40 years | Blood, bone marrow |
Body 3 | Adult, female, age> 55 years | Blood, bone marrow |
Body 4 | Child, male, age> 6 years | Bone marrow |
Body 5 | Adult, female, age> 55 years | Bone marrow |
Body 6 | Adult, male, age 20-40 years | Blood, bone marrow |
Body 7 | Adult, female, age 20-40 years | Bone marrow |
Body 8 | Child, female, age 10-12 years | Bone marrow |
Body 9 | Adult, female, age 20-40 years | Bone marrow, muscle |
Body 10 | Adult, male, middle-aged to elderly | Blood, bone marrow |
Body 11 | Adult, female, age 20-40 years | Bone marrow, muscle |
Body 12 | Adult, male, age 20-40 years | Blood |
Body 13 | Adult, male, middle-aged to elderly | Blood, bone marrow |
Table 2 shows the Hae III allele sizes (in base pairs) obtained at the genetic loci D2S44, D4S139, D5S110, and D10S28 from the biological samples of the 13 victims.
Victims |
D2S44 (YNH24) |
D4S139 (PH30) |
D5S110 (LH1) |
D10S28 (TBQ7) |
Body 1* | 2636 | 5266 | 2035 | 1737 |
2243 | 3911 | 1365 | 1012 | |
Body 2 | 2060 | 5271 | 3125 | 3271 |
1456 | 4483 | 2186 | 1562 | |
Body 3 | 1921 | 8684 | 5626 | 2119 |
1790 | 5544 | 1513 | 1215 | |
Body 4 | 1661 | 8727 | 5593 | 2102 |
1519 | 7734 | 3445 | 977 | |
Body 5 | 1695 | 9060 | 5239 | 2255 |
1405 | 3039 | 2421 | 1683 | |
Body 6 | 1928 | 5131 | 4357 | 2905 |
1035 | 3230 | 1987 | 1165 | |
Body 7 | 2101 | 6933 | 7185 | 4583 |
1988 | 5841 | 4583 | 1015 | |
Body 8* | 2617 | 5231 | 2010 | 1723 |
2225 | 3888 | 1351 | 1009 | |
Body 9 | 1785 | 8630 | 5551 | 2088 |
1524 | 6823 | 2161 | 1207 | |
Body 10 | 1819 | 7861 | 3037 | 1548 |
1762 | 6034 | 1571 | 968 | |
Body 11 | 1910 | 7488 | 2980 | 1733 |
1789 | 4644 | 2719 | 970 | |
Body 12 | 2719 | 7488 | 3436 | 967 |
1636 | 6633 | 3076 | 653 | |
Body 13 | 1876 | 9270 | 2991 | 1752 |
1461 | 5317 | 1336 | 959 | |
* Body 1 and Body 8 have matching alleles at all four genetic loci, indicating that these individuals were identical twins. This determination was consistent with the information obtained from their next of kin. |
Figures 1 through 6 show the family trees of the six different families deduced from the reference blood samples. All 13 bodies of the victims were subsequently identified from these family trees.
Table 3 summarizes the findings. The match window used by the DNA laboratory is ± 2.5 percent (i.e., the maximum size difference between the two matching alleles is 5 percent). Comparisons were carried out using the fixed-bin analysis, and some of them involved intergel comparisons (Budowle et al. 1991; Morris et al. 1989; Endean 1989).
Family Number | Body Number |
1 | 2 |
2 | 6 |
3 | 5 |
4 | 1, 8, and 13 |
5 | 7, 10, and 11 |
6 | 3, 4, 9, and 12 |
In comparing all of the results, no homozygous alleles were found in any of the 13 victims, and no adult male or female profiles were similar enough to be confused or matched with any other reference donors. This closed system was relatively straightforward for identification by elimination.
Note that, in Figure 4, the results of the D4S139 marker were not obtained for the daughter. One of the reasons could be that this marker was the last to be developed. After hybridization and stripping for the three other markers, it is possible that there was insufficient DNA for this final hybridization. Another factor was time. Most of the families were waiting anxiously to collect the remains of their relatives and there was little time available for a repeat of the entire RFLP process, which can take up to a week. Despite the unavailability of results for this marker, however, the identities of Bodies 1, 8, and 13 were still ascertained positively.
This case, the first of its nature to be handled by the DNA laboratory, provided several valuable lessons:
Budowle, B., Waye, J. S., Shutler, G. G., and Baechtel, F. S. Hae III-A suitable restriction endonuclease for restriction fragment length polymorphism analysis of biological evidence samples, Journal of Forensic Sciences (1990) 35:530-536.
Budowle, B., Giusti, A. M., Waye, J. S., Baechtel, F. S., Fourney, R. M., Adams, D. E., Presley, L. A., Deadman, H. A., and Monson, K. L. Fixed-bin analysis for statistical evaluation of continuous distributions of allelic data from VNTR loci, for use in forensic comparisons, American Journal of Human Genetics (1991) 48:841-855.
Morris, J. W., Sanda, A. I., and Glassberg, J. Biostatistical evaluation of evidence from continuous allele frequency distribution deoxyribonucleic acid (DNA) probes in reference to disputed paternity and identity, Journal of Forensic Sciences (1989) 34:1311-1317.
FBI Laboratory. Procedures for the detection of restriction fragment length polymorphism in human DNA, Federal Bureau of Investigation, December 1990.
Endean, D. J. RFLP analysis for paternity testing: Observations and caveats. In: The International Symposium on Human Identification. Promega Corporation, Madison, Wisconsin, 1989, pp. 55-76.
Home Office Forensic Science Services. The analysis of samples for DNA profiling (version 2). Central Research and Support Establishment, Home Office Forensic Science Services, Aldermaston, Reading, Berkshire, Great Britain, March 1990.
FORENSIC SCIENCE COMMUNICATIONS JULY 2000 VOLUME 2 NUMBER 3
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