In Malaysia, as well as other forensic laboratories in tropical climates many of the crime scene samples received at the forensic laboratory are less than ideal. They are often present low amounts and/or degraded due to environmental exposure to high temperatures, sun and humidity for days or even months. STR analysis is widely accepted by forensic community, but sometimes this technique gives unreliable results when profiling degraded samples as the amplicons size are relatively larger (100 bp to 450 bp). While, miniSTR is a reduced size of STR amplicons which enables higher recovery of information from degraded samples, but only a few loci are amplified and allele drop out still may occur, as the amplicons are up to 200 bp. The percentage of recovery DNA profile from degraded DNA using mtDNA is much higher due to its present in cells at a much higher copy number than the nuclear DNA. However, the major drawback for mtDNA is labour intensive and has a low information value (i.e. it is not highly discriminating). Insertion/deletion polymorphisms (INDELs) are relatively new class of a DNA marker used in forensic casework; used most commonly as a supplementary method to STR (Short Tandem Repeat) based typing. INDELs, like SNPs (Single Nucleotide Polymorphisms), are particularly useful for the analysis of highly degraded DNA as the amplicon sizes are typically below 160 bp; they can also be valuable as an additional tool to help resolve kinship cases, with the advantage over STRs that do not have high mutation rates. INDELs have an advantage over SNPs in that they are length polymorphisms and so can be analysed by simply measuring the length of the allele(s). The Qiagen Investigator DIPplex® kit is currently one of two commercially available kits for the amplification of INDEL polymorphisms; it amplifies 30 biallellic INDEL loci and the amelogenin locus. The primers used are fluorescence labelled with 6-FAM, BTG, BTY and BTR. This technique is robust, relatively simple, and the results are analysed using the same capillary electrophoresis equipment and software as used for STR typing. The INDEL markers have simple biallelic structure and combine the advantages of STR and SNP assays. This study has established that the INDEL technique, using the Investigator® DIPplex PCR kit, is a simple, informative and sensitive approach for the typing of degraded DNA, as compared to STRs and SNPs. In this research, allele frequencies for 30 autosomal INDEL loci were studied in 500 unrelated individuals (100 each) from Malay, Malay-Chinese (M-Chinese), Malay-Indian (M-Indians), Iban and Bidayuh. The PCR amplification used the Qiagen Investigator® DIPplex kit. These population groups represent the majority of the population in Malaysia. No significant departure from Hardy Weinberg Equilibrium (HWE) expectations were observed for most of the INDEL loci analyzed (p-value >0.05) on the Malaysian population samples. The exceptions were HLD101 for Malay (p = 0.0009), HLD133 for M-Indian (p=0.005), HLD125 for Iban (p=0.028) and HLD93 for Bidayuh (p = 0.014). However, when the Bonferroni correction for multiple testing performed on the population samples, none of the previous p-values was significant. There were no Malaysian population studies was carried out using the Qiagen Investigator® Investigator DIPplex kit at the time of the research. This INDEL assay have undergo an extensive valdidation process and novelty report of the allele frequencies of INDELs would serve as reference database for individual identification in the Malaysian population in the future. Even the match probability of the STR is higher, INDEL still gives an acceptable value for forensic identification; e.g. linking different pieces of evidence or re-association of body parts in the case of human identification. Biological samples received in Malaysia forensic laboratory have often been exposed to unfavourable environmental conditions. This can lead to DNA degradation and end up in incomplete DNA profiles. It is difficult to distinguish between low template DNA that are producing no or partial profiles because of DNA degradation and those that produce no or incomplete profile because of PCR inhibition. Even though real-time PCR methods are available for quantification and detection of PCR inhibitors, the information received is limited as real-time PCR targets amplicons that are much smaller than those typically targeted in forensic analysis. To gain more information on the quality of extracted DNA, a new multiplex PCR assay comprising a Mini 4-plex targeting amplicons of 50 base pairs (bp), 70 bp, 112 bp and 154 bp along with two Internal Amplification Controls (IACs) of 90 bp and 170 bp was developed. The primers were redesigned from a 4 plex & IACs system developed by previous PhD UCLan students. This multiplex was optimised so that it worked efficiently on DNA template as low as 0.009 ng, which highlighted the strength of the Mini 4-plex system. The IACs were effective in detecting PCR inhibitors. The Mini 4-plex system (Mini 4-plex & IACs) was demonstrated to be an effective tool for identifying degraded and inhibited samples, which could be used to triage forensic samples in a casework laboratory. Therefore, this study has led to the improvement of new and novel markers assessing DNA degradation and PCR inhibition on forensic samples. This will demonstrate the compatibility with forensic laboratory workflows. The need of this Mini 4-plex assay in forensic laboratory can reduce time and cost of DNA analysis. Besides it will contribute to a good management samples, where after being assessed the samples can be decided to analyse using appropriate kit (e.g. miniFiler or INDEL). Indirectly, this will increase the quality of the sample itself. In order to increase the power of a 15 Mini-INDEL multiplex, which was developed earlier by UCLan PhD student, a total of 9 autosomal INDEL markers that are not part of the Qiagen Investigator DIPplex® kit were selected and redesigned from (Pereira et al. 2009). In this study a simple and sensitive INDEL multiplex was successfully developed for human identification. However, the discrimination power is still low when compared to STR systems, but has potential value when analysing highly degraded material. By combining the 15 Mini-INDELs and 9 Mini-INDELs allele frequency data, it will give beneficial by increasing the match probability values in future analysis.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:727089 |
| Date | January 2017 |
| Creators | Hassan, Nur Haliza Binti |
| Publisher | University of Central Lancashire |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://clok.uclan.ac.uk/20673/ |
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