Short Tandem Repeat (STR) Data for the AmpF/STRa Profiler Loci of Samples from the Malay Population in Malaysia
April 2002 - Volume 4 - Number 2
Research and Technology
Short Tandem Repeat (STR) Data for the AmpFlSTRâ Profiler Loci of Samples from the Malay Population in Malaysia
Lim Kong Boon
Mohd Izuan Othman
N. Hithaya Jeevan
Head of Serology/DNA Section
Kee Sue Sing
Director of the Forensic Division
Lee Yoon Hin
Assistant Scientific Officer
Department of Chemistry
Ministry of Science, Technology, and the Environment
The collection and analysis of DNA from an ethnic population can be useful for forensic evaluation. In this study, allele frequencies were generated for the nine short tandem repeat (STR) genetic loci included in the AmpFlSTRâ Profilerä Amplification Kit (PE Applied Biosystems, Foster City, California). These frequencies were compared between two groups of blood samples from unrelated individuals of the Malaysian Malay population. The first group of blood samples was acquired from blood banks of various hospitals, whereas the second group was acquired from blood samples submitted for casework analysis. The combined power of discrimination and mean paternity exclusion probability of the nineplex was greater than 99.99999996 percent and 99.93 percent, respectively for the two different groups. Only one of the nine loci, CSF1PO of the blood bank samples (p=0.0087), deviated from Hardy-Weinberg Equilibrium expectations using the exact test. Upon comparison of the two blood sample groups, there were no significant differences among their statistical estimates, and the scatter plot demonstrated the similarity between the groups. This survey increased the database’s STR data for the AmpFlSTRâ Profiler loci of samples from the Malay population in Malaysia.
The collection and analysis of DNA from an ethnic population can be useful for forensic evaluation. Malaysia is a multiracial country comprised mainly of three population-ethnic groups — Malay, Chinese, and Indian. The Malays comprise about 60 percent of the population. STR allele frequencies for the AmpFlSTRâ Profiler loci from these three ethnic population groups were compiled using samples of blood from Malaysians who were donors to blood banks of various hospitals for the statistical evaluation of forensic DNA evidence (National Research Council 1996; Technical Working Groups on DNA Analysis Methods 1995). Also DNA profiles of Malaysians whose blood samples were submitted for casework during 1998-2000 were compiled. The data from both groups were pooled in the database, increasing the size of the database for comparison and evaluation.
Samples of blood from Malay donors were acquired from the blood blanks of various hospitals for forensic DNA evaluation. A total of 139 to156 samples of this group were used. Samples were also used from 217 to 220 Malays whose blood samples were submitted for casework during 1998-2000. DNA was extracted from whole blood from the blood bank samples with phenol/chloroform/isoamyl alcohol after proteinase K digestion (PE Applied Biosystems 1995, 1996A, 1996B, 1997). Quantifying was done using agarose minigel electrophoresis with lambda DNA as the standard.
DNA was also extracted from the casework whole blood samples using the chelex extraction protocol (PE Applied Biosystems 1995, 1996A, 1996B, 1997) and quantified using the Quanti-Blot technique. About 1-2 ng of DNA were used for each amplification, which was done in a thin-walled 0.2 ml PCR reaction tube in a GeneAmpâ PCR System (PE Applied Biosystems, Foster City, California). The thermal cycling parameters were as follows: hold at 95oC for 11 minutes, followed by 28 cycles of 94oC for 1 minute, 59oC for 1 minute, 72oC for 1 minute, then hold at 60oC for 45 minutes. Polyacrylamide gel electrophoresis and detection of alleles were performed using the 377 DNA Sequencer with the GeneScanâ Analysis and GenoTyperâ software (PE Applied Biosystems, Foster City, California) (Anderson et al. 1996; Schumm et al. 1998).
Allele frequencies were calculated for each STR locus, and the number of heterozygotes, both observed and expected (Hobs. and Hexp.), was determined (Chakraborty et al. 1993). The Hardy-Weinberg Equilibrium was verified using the exact test (P) (Brenner 2000). Only one of the nine loci, CSF1PO of the blood bank samples (p=0.0087), deviated from Hardy-Weinberg Equilibrium expectations. Power of discrimination (PD) and mean paternity exclusion probability (MEP) were calculated for each locus and for the combined nine STR loci (Brenner and Morris 1989; Morgan and Cox 1995). The combined power of discrimination and mean paternity exclusion probability of the nineplex was greater than 99.99999996 percent and 99.93 percent, respectively for the two different groups. Ratios of the maximum-to-minimum frequency estimates and the scatter-plot approach were evaluated using 145 different DNA profiles obtained from crime samples (Budowle et al. 1994A; Budowle et al. 1994B). Upon comparison of the two blood sample groups, there were no significant differences among their statistical estimates, and the scatter plot demonstrated the similarity between the groups.
- Differences in relative allele frequency vary from less than 0.0100 (for 49 of the total 84 alleles detected) to a high of 0.0846 (vWA-allele 14). A summary of the differences is shown in Table 4.
- Observed and expected heterozygosity vary from 0.0040 (CSF1PO) to 0.0784 (THO1) and from 0.0004 (TPOX) to 0.0287 (FGA), respectively.
- Power of discrimination and mean paternity exclusion probability vary from 0.0005 (FGA) to 0.0146 (THO1) and from 0.0026 (D13S317) to 0.0327 (THO1), respectively.
For the nine genetic loci analyzed, there were only four alleles out of the total of 84 alleles detected showing differences in relative allelic frequencies greater than 0.05. They were FGA-allele 23 (0.0517), THO1-allele 7 (0.0534), D5S818-allele 10 (0.0737), and vWA-allele 14 (0.0846).
Ratios in Table 5 were determined by dividing the largest frequency by the smallest frequency for each of the 145 crime DNA profile samples.
Most of the 145 DNA profiles used consisted of 9 genetic loci, whereas there were 6 eight-loci profiles, 5 seven-loci profiles, and 2 six-loci profiles.
More than 84 percent of the frequency estimates between the databases of the blood bank and casework samples had differences of less than 3 fold and less than 3 percent more than 5 fold (maximum difference of about 6.3 fold). Therefore, there were no significant differences among their statistical estimates using the databases from the two different groups.
|Figure 1 Scatter Plot Comparison of the Frequency Estimates of 145 Crime DNA Profile Samples Between the Databases of the Blood Bank and Casework Samples. Click to enlarge image.|
The breadth of the scatter plot (Figure 1) is narrow, demonstrating the similarity of the databases from the two different blood sample groups. Data from both groups were combined to constitute the Malaysian Malay population database for the statistical evaluation of DNA evidence.
Anderson, J. F., Greenhalgh, M. J., Butler, H. R., Kilpatrick, S. R., Piercy, R. C., Way, K. A., Myhill, H. S., Wright, J. C., Hallett, R., and Parkin, B. H. Further validation of a multiplex STR system for use in routine forensic identity testing, Forensic Science International (1996) 78:47-64.
Brenner, C. and Morris, J. Paternity index calculation in single locus hypervariable DNA probes: Validation and other studies. In: Proceedings of the International Symposium on Human Identification, Memorial Genetics Center, Long Beach, California, 1989, pp. 21-53.
Brenner, C. DNA.VIEWTM, DNA Forensic Analysis Programs, User’s Manual. Consulting in Forensic Mathematics, Oakland, California, 2000.
Budowle, B., Monson, K. L., Giusti, A. M., and Brown, B. Assessment of frequency estimates of Hae III-generated VNTR profiles in various reference databases, Journal of Forensic Sciences (1994A) 39(2):319-352.
Budowle, B., Monson, K. L., Giusti, A. M., and Brown, B. Evaluation of Hinf I-generated VNTR profile frequencies determined using various ethnic databases, Journal of Forensic Sciences (1994B) 39(4):988-1008.
Chakraborty, R., Srinivasan, M., and Daiger, S. P. Evaluation of standard error and confidence interval of estimated genotype probabilities and their implication in DNA forensics, American Journal of Human Genetics (1993) 52:60-70.
Morgan, G. R. and Cox, K. J. Allele and Genotype Analysis: Statistical Package (Version 2.23) Geoffrey Morgan, Queensland, Australia, 1995.
National Research Council. The Evaluation of Forensic DNA Evidence, National Academy Press, Washington, DC, 1996.
PE Applied Biosystems. ABI Prism 377 DNA Sequencer. User’s Manual. PE Applied Biosystems, Foster City, California, 1995.
PE Applied Biosystems. ABI Prism Genotyper 2.0. User’s Manual. PE Applied Biosystems, Foster City, California, 1996A.
PE Applied Biosystems. ABI Prism GeneScan Analysis Software. User’s Manual. PE Applied Biosystems, Foster City, California, 1996B.
PE Applied Biosystems. AmpF/STRâ Profiler PCR Amplification Kit. User’s Manual. PE Applied Biosystems, Foster City, California, 1997.
Schumm, J. W., Sprecher, C. J., Lins, A. M., Micka, K. A., Rabbach, D. R., Taylor, J. A., and Bacher, J. W. Automated fluorescent detection of 8-locus and 4-locus STR multiplexes, Progress in Forensic Genetics (1998) 7: 43-45.
Technical Working Group on DNA Analysis Methods. Guidelines for a quality assurance program for DNA analysis, Crime Laboratory Digest (1995) 22: 21-43.