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Identification of Severely Burned Bodies From a Vehicle Collision Using the Restriction Fragment Length Polymorphism (RFLP) Technique, by Lim, Jaya, Kassim, Seah, Lee, and Chee (Forensic Science Communications, July 2000)

Identification of Severely Burned Bodies From a Vehicle Collision Using the Restriction Fragment Length Polymorphism (RFLP) Technique, by Lim, Jaya, Kassim, Seah, Lee, and Chee (Forensic Science Communications, July 2000)

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July 2000 - Volume 2 - Number 3

Case Report:

Identification of Severely Burned Bodies From a Vehicle Collision Using the Restriction Fragment Length Polymorphism (RFLP) Technique

Kong Boon Lim Primulapathi Jaya, Baktiar Kassim,
and Lay Hong Seah

Scientific Officers, Department of Chemistry
Ministry of Science, Technology, and the Environment
Malaysia

Yoon Hin Lee
Technical Officer, Department of Chemistry
Ministry of Science, Technology, and the Environment
Malaysia

Ong Koh Chee
Former Director of Forensic Division, Department of Chemistry
Ministry of Science, Technology, and the Environment
Malaysia

| Abstract | Introduction
Materials and Methods Results and Discussion
References | Figures

Abstract

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.

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Introduction

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.

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Material and Methods

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 loci—D2S44, D4S139, D5S110, and D10S28—using 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.

Table 1

Physical Identities of the Accident Victims and Types
of Samples Submitted for DNA Analysis

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

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Results and Discussion

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.

Table 2

Alleles Detected in Biological Samples
from the 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.

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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).

Table 3

Summary of the Findings

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


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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:

  • The interpretation of results should be finalized and confirmed only after all results from the victims and reference donors have been obtained and studied together.

  • Good coordination between DNA analysts and forensic pathologists will enhance the identification process.

  • Cooperation between governments is essential in facilitating the collection of reference samples.

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References

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.

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