Methods

3.3.1. Samples

The samples used in this study were blood stains on FTA® cards, obtained from five

Arab individuals from Kuwait, and blood stains on five cotton swatches, obtained from

five Arab individuals from the UAE.

The purpose of including these two Arab populations was to generate in-house SNP

data that could be used to identify informative SNPs for forensic purposes. Also, when

this study was conducted, it was the first time that samples from UAE and Kuwaiti

individuals had been used in this type of investigation.

3.3.1.1. DNA Extraction and Quantification

Extraction of DNA from the 10 samples was performed using a standard

phenol/chloroform procedure following digestion with Proteinase K as described in

Section 2.2.1.1. This method was selected in order to achieve a high yield of DNA

template (Dixon et al., 2005a). Following extraction, the concentration of DNA was

estimated using the Quantifiler® Human DNA Quantification kit (Applied Biosystems)

with the ABI 7500 real-time PCR. Samples with insufficient concentrations (< 50 ng/µl)

were amplified using phi 29 DNA polymerase, as described in Section 2.2.5. The

extraction and quantification of samples was carried out as described in Sections 2.2.1.1

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3.3.2. Genotyping Methods and Techniques

3.3.2.1. Affymetrix

GeneChip

Technique

Allele Specific Hybridisation Method

Allele specific hybridisation is the basis of the Affymetrix GeneChip® system (Figure

3.2). This method is based on the annealing of a labelled amplicon containing the

polymorphic site to a probe that is attached to an array (Goto et al., 2002). Annealing

occurs as the amplicon contains the complementary sequence to the probe (Wallace et

al., 1979). The hybridisation reaction is washed to remove any mismatch strands,

enabling the complementary strands to be detected.

Probe

Figure 3.2. Shown above is an illustration of the allele specific hybridisation method. [A] represents a biotinylated single strand amplicon which hybridises perfectly with the complementary probe sequence to form a stable double strand; [B] represents a mismatch double strand which is removed during the post-hybridisation wash.

GeneChip® Method

The main feature of GeneChip® is the capability to detect thousands of SNPs in a single reaction. Each microarray contains sets of DNA probes with the SNP sequences that were selected from GenBank®. These probes are designed to be sensitive and specifically to hybridise only to the target sequence (Liu et al., 2003). In this project GeneChip® Mapping 250K Arrays Sty kit was used (Figure 3.3).

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In this method there were three main steps: (1) PCR amplification of the DNA sequence

containing the target SNP; (2) fragmentation of PCR products using endonuclease

DNase І; (3) labelling of PCR products and hybridisation to the probes in the arrays (Figure 3.4).

Genomic DNA (250 ng) was digested using the restriction enzyme Sty which cut the

target DNA into segments that were, on average, between 250 bp and 1,000 bp. The

digested fragments become the substrate for the adapter ligation enzyme which attached

an adapter. A single common primer, complementary to the adapter, was used to

amplify the fragments (Matsuzaki et al., 2004). The PCR products were then

fragmented by the enzyme DNase І. Finally, the fragments were biotinylated before

hybridisation to the array probes by allele specific hybridisation. Subsequently, only the

complementary sequences attached to array probes would be detected after purification

and staining with Streptavidin Phycoerythrin. Genotyping Analysis Software (GTYPE)

and GeneChip® Operating software (GCOS) were used for SNP detection.

Front Back

Plastic cartridge

Probe array on glass substrate

Figure 3.3. Shown above is the Affymetrix® GeneChip® Probe Array consisting of a square glass substrate mounted in a plastic cartridge. The glass contains an array of oligonucleotides mounted on its inner surface.

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Sty Sty Sty

DNA strand

Sty Digestion

Ligation

One primer Amplification

Fragmentation & Biotin Labelling

Hybridization and Detection

Figure 3.4. Shown above is the digestion of human genomic DNA with Sty and then the ligation of an adapter which contains a PCR primer site. The DNA is amplified, using the common primer, and the fragments are then digested by DNAse І to an average size of less than 180 bp, labelled with biotin, and then hybridised to the GeneChip® Mapping 250K Array. Figure 3.4 was adapted from Matsuzaki et al. (2004).

3.3.2.2. Strategies and Criteria for SNPs Selection

In order to obtain informative SNP markers, strategies and criteria were formulated.

Based on the previous strategies that were described in Section 2.3.4, the selection of

100 SNPs as an initial target was carried out. The number of SNPs selected on each

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SNPs from Y and X chromosomes where eliminated from the selection, profiles of

autosomal SNP exhibit high variability due to chromosomal assortment recombination

and mutation leading to low match probability (Jobling and Gill, 2004). Y-chromosome

is male specific and less diverse than autosomal SNPs as mutation is the only function

to diversity for the Y haplotypes, therefore Y profiles show relatively high match

probability (Jobling and Gill, 2004). Profiles from X chromosome showed less variation

from the autosomal profiles, this due to low heterozygosity level on X chromosome;

possibly due to strong selection on the X chromosome which is owing to the

hemizgosity in male (Sachidanandam, et al., 2001).

Table 3.1 Shown below are the different number of SNPs that were selected on each autosomal chromosome in the genome. The target number of SNPs selected was based on the size of each chromosome. Chromosome length was obtained from Ensembl Genome Browser (www.ensembl.org).

Chromosome Chromosome size (Mb) Percentage (Mb%) Target number of SNPs 1 247 8.6 9 2 243 8.5 9 3 200 7.0 7 4 191 6.7 7 5 181 6.3 6 6 171 6.0 6 7 159 5.5 5 8 146 5.1 5 9 140 4.9 5 10 135 4.7 5 11 134 4.7 5 12 132 4.6 5 13 114 4.0 4 14 106 3.7 4 15 100 3.5 3 16 89 3.1 3 17 79 2.8 3 18 76 2.7 3 19 64 2.2 2 20 62 2.2 2 21 46.9 1.6 1 22 50 1.7 1 Total 2866 100 100

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