عنوان

Single nucleotide polymorphisms :

TLets565897

Single nucleotide polymorphisms :

[Thesis]

Sanqoor, Shaikha Hassan

characterisation and application to profiling of degraded DNA

University of Central Lancashire

2009

Thesis (Ph.D.)

2009

Single nucleotide polymorphisms (SNPs) are one of the forensic markers used to resolve the problem of DNA typing from degraded samples. It has been found in previous studies that when profiling heavily degraded forensic samples the small amplicon required for SNP analysis has an advantage over the larger STR loci, which are routinely used in forensic case work. A total of 66 SNPs from the non-coding region of the 22 pairs of autosomal chromosomes were identified and SNP assays developed. Instead of selecting the SNPs from the available GenBank® sites, SNPs were typed from Arab individuals from Kuwait and United Arab Emirates (UAE) to identify polymorphic SNPs. In order to obtain SNP data from Arab populations, a total of 10 unrelated Arab individuals from Kuwait and UAE were typed. The Affymetrix GeneChip® Mapping 250 K Array Sty І was employed to generate profiles for approximately 238,000 SNPs. Only autosomal SNPs were selected from the data. Following selection, allele frequencies were estimated using the SNaPshot™ technique (Applied Biosystems) with 25 UAE individuals. For this technique, PCR forward and reverse primers were designed to generate PCR products less than 150 bp. The single base extension primers were designed to hybridise 1 bp upstream from the target SNP. SNP characterization, including Hardy¬Weinberg equilibrium and pair wise linkage disequilibrium, was carried out using the software package Arlequin v 3.1. Allele frequencies were calculated using Excel spreadsheets. PowerStats v.12 software used for discrimination power and match probability estimation. All the 66 SNPs were polymorphic with average heterozygosity levels of 47%. A high heterozygosity level is very valuable for forensic application improving the individualization of forensic samples (Vallone et al. 2005). The probability that two individuals having identical genotype profile was found to be very low, 3.058 x 10-25. The combined power of discrimination was found to be 0.999999999. This indicated that the selected SNPs met the parameters needed for forensic application. The SNPs genotype sensitivity gave profiles from minute amounts of DNA template as little as 100 pico grams (pg) and optimal and reproducible results at 300 pg of DNA template. The profiling of DNA from forensic samples is not always possible. This can be due to insufficient amount of samples being recovered and in many cases, DNA degradation. Biological materials that are recovered from the scene of the crime have often been exposed to sub-optimal environmental conditions such as high temperature and humidity. SNPs performance on degraded samples was tested on artificially degraded saliva and semen samples. Controlled temperature and humidity experiments were performed to study the effect of these environmental factors on the samples. Also uncontrolled experiments on samples being subjected to different weather conditions (UK summer and UAE winter and summer) was performed in order to study and compare both weather effects on saliva samples. The triplex sets of SNPs that were developed for such study showed full allele profiles when compared to STRs, the current method used in forensic labs. In addition, SNPs produced a higher success rate than STRs when tested with samples obtained from human teeth remains and on samples subjected to DNase 1 digestion. The small size of SNPs, between 90 and 147 base pair (bp), showed more resistance to degradation than the STRs size ranging between 100 and 360 bp. This study demonstrated that the 66 SNPs selected are useful markers when the typing of degraded samples by STRs fails to produce complete or partial profiles.

QH426 Genetics ; QR Microbiology ; RA1001 Forensic Medicine. Medical jurisprudence. Legal medicine

Sanqoor, Shaikha Hassan

University of Central Lancashire

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[Thesis]

276903

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