Summary

Application of autosomal (AS) and Y-chromosomal STR markers as well as mitochondrial markers (mtDNA) for kinship and forensic (paternity, maternity and other family relationships) investigation is in routine practice now in Sri Lanka. However there are instances of kinship & forensic importance, which cannot be entirely resolved using these methods. X-chromosome STR (X-STR) analysis could complement autosomal and Y chromosome STR analysis at such instances. However, in order to use X-STR markers for any case work, appropriate X-STR markers for the Sri Lankan population has to be first identified and the allelic and haplotype frequencies of these selected X-STRs markers has to be find out. This needs establishing a database comprising of both allele and haploytype frequencies of an appropriate set of X-STR markers for Sri Lankan populations, a requirement that had long been recognized by the forensic community. Nevertheless, no attempt has been made so far in filling this need, perhaps due to the laborious nature of the study. However with the ever-increasing crime rates, divorce cases, child adoption, migration events and natural disasters, there is now a pressing need for immediate intervention.

Accordingly, the proposed study try to address this requirement by establishing a comprehensive database for X-STR markers among Sri Lankan population covering all ethnic groups. Initially 16 X Chromosomal STR loci which are highly polymorphic and widely used by the international forensic community will be selected based on published literature to conduct a pilot study with 30 individual from each of the main ethnic groups in Sri Lanka. Based on the results obtained from this study, most appropriate 12 X-STR markers for Sri Lankan population will be selected and an in-house single reaction X-STR multiplex PCR assay for all 12 markers, tailor-made for Sri Lankan populations will be developed. Using this assay, a total of 500 individuals representing all four ethnic groups will be genotyped for the 12 selected X-STR markers to construct a comprehensive X-STR database consisting of allele and haplotype frequencies. The applicability of the newly developed assay and the database in resolving complex kinship/forensic scenarios will then be tested with actual case studies.

However, a 12 marker multiplex assay will be quite expensive due to automated analysis required at the DNA typing phase, and not be affordable for the use by general public, although it is quite cost effective for a large population study, as the one described above. Hence, for the routine use by the Sri Lankan forensic community, a more affordable multiplex assays (three separate multiplex assays each comprising of four markers or two multiplex assays each covering six marker) will be developed. Due to the less complex nature of these assays, manual polyacrylamide gel electrophoresis (PAGE) will be sufficient for allele resolution of these assays, compared to the automated capillary gel electrophoresis required for the single reaction 12 X-STR multiplex. Thus, the project at the end will develop two different assays (one automated and one manual) to be used according to the requirement of the user.

As such, this study will immensely benefit the general public and Sri Lankan Judiciary, CID etc. enabling the resolution of kinship and forensic cases that cannot be conclusively resolved at present, using the methods available such as autosomal and Y chromosome STR analysis and mitochondrial marker analysis.

Objectives

To construct a comprehensive X-STR database consisting of allele and haplotype frequencies to be used in kinship and forensic case work in Sri Lanka and to test its applicability using case studies. This will be carried out in three steps;

1. Identifying the most appropriate X-STR markers for the different ethnic groups in Sri Lanka

2. Developing an in-house X-STR assay, tailor-made for Sri Lankan populations

3. Testing the applicability of this assay and the database in resolving complex kinship/forensic scenarios using actual case studies.

A more detailed list of specific objectives is given below;

1. To conduct a pilot study to estimate the population genetic parameters of 16 X-STR loci (the commonly used markers based on literature) among Sinhalese (n=30), Sri Lankan Tamil (n=30), Indian Tamil (n=30) and Sri Lankan Moor (n=30) populations in Sri Lanka.

2. To select 12 most appropriate X-STR loci for Sri Lankan population based on the results obtained in above objective 1.

3. To design allelic ladders for each selected X-STR marker using a mixture of samples that represent each allele observed in the population dataset.

4. To develop an in-house multiplex PCR assays (suitable for automated analysis) to investigate the selected 12 X-STR markers.

5. To establish a comprehensive allele frequency database of Sinhalese, Sri Lankan Tamil, Indian Tamil and Sri Lankan Moor populations with respect to 12 X-STR loci using total of 500 individuals

6. To establish the X-STR haplotype and other population genetic parameters with respect to the four different ethnic groups in Sri Lankan population

7. To assess the genetic diversity of the four ethnic groups above with respect to the 12 X-STR markers

8. To check the applicability of the newly developed PCR assay and the X-STR frequency database using actual kinship and forensic cases

9. To develop a low cost, affordable X-STR multiplex PCR assay in 2-3 reactions for manual DNA typing thorough polyacrylamide gel electrophoresis PAGE to allow integration with the routine DNA casework done in Sri Lanka

Major Equipment Facilitated by Grant