Materials & Methods

2.4.1 DNA Extraction from peripheral lymphocytes

Approximately 5 ml of blood was collected into EDTA vacutainers (Beckton Dickenson). DNA was extracted from whole blood with the QIAamp Blood Kit (Qiagen), using the “large sample volume blood and body fluid protocol”.

EDTA blood (1 ml) was incubated for 10 min at 70 C with 1 ml of lysis buffer (AL) and 125

l of proteinase K, after which 1050 l of 100% ethanol was added. The sample was then passed through a Qiagen spin column by centrifugation (at 8000 rpm for 1 min) in a Mikro 20 Patterson Scientific centrifuge. The column was washed twice with washing buffer (AW), prior to the elution of DNA by centrifugation (at 8000rpm for 1 min), with 200 l of elution buffer (AE) preheated to 70 C.

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2.4.2 DNA quantification by spectrophotometer

DNA was quantified by measurement of absorbance at 260 nm wavelength () using a Gene Quant pro spectrophotometer (Amersham Pharmacia Biotech). DNA concentration was calculated based on a solution of 50 g/ml having an optical density of 1 at 260 nm, and with a light path of 1cm. An absorbance ratio 260/280 of greater than 1.8 is indicative of a pure DNA sample.

2.4.3 Genomic DNA Preparation

Working stocks of all DNA samples were prepared to be 50ng/μl, as this concentration is required by the protocol for DNA digestion step, for high throughput assay, TE buffer (0.1 mM EDTA, 10 mM Tris HCl, pH 8.0) was used for the dilution process when required. Five μl (50ng/μl) of each diluted genomic DNA has been aliquoted into each well of the 96-well plate.

2.4.4 DNA control

The Affymetrix kit has provided DNA reference control (reference genomic DNA 103), to be processed with the samples as part of quality control check. Thus that DNA was also processed with DNA of family member.

2.4.5 DNA digestion with Xba1 restriction enzymes

A master mix of 10.5 μl H2O, 2 μl of NE buffer 2 (10X), 2 μl of bovine serum albumin (BSA) (10X (1mg/ml)) and 0.5μl of Xba1 (20 u/μl) was prepared on ice. Fifteen μl of the master mix was then added to the 5 μl (50ng/μl) of the diluted genomic DNA in the 96-well plate. The plate has been covered with a plate cover and sealed tightly, mixed by vortex, and centrifuged briefly at 2,000 rpm for 1 minute. The plate was then placed in a thermal cycler for 120 minutes at 37 C and hold at 4 C.

2.4.6 DNA Ligation

A master mix of 1.25 µ of (5μM) Adaptor Xba and 2.5µ T4 DNA Ligase buffer (10X) was prepared. In each digested DNA sample, 3.75 μl of the Ligation Master Mix has been added followed by 1.25 μl of T4 DNA Ligase making the final reaction volume 25µl. The plate was then covered with a plate cover and sealed tightly, vortexed at medium speed for 2 seconds, and spun briefly at 2,000 rpm, for 1 minute. The plate was then placed in a thermal cycler (at 16 C for 120 minutes then 70 C for 20 minutes). Each DNA ligation reaction has been diluted

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by adding 75 μl of molecular biology-grade H2O to make the final volume of ligated DNA to be 100µl.

2.4.7 PCR

PCR Master Mix

2.4.7.1

A master mix was prepared in the pre-PCR clean room; it comprised 10 μl of PCR buffer (10X), 10µl of dNTP (2.5 mM each), 10µl of MgCl2 (25 mM), 7.5µl of PCR Primer Xba (10 μM), 2 μl AmpliTaq Gold® (5 U/μl) and 50.5µl of H2O making the total of 90µl.

PCR staging

2.4.7.2

PCR master mix (90 μl) was added to each well of a new PCR plate, followed by diluted, ligated DNA (10 μl) with 4 wells being used per sample. The plate was then sealed with plate cover, vortexed at medium speed for 2 seconds and spun at 2,000 rpm for 1 minute. The plate was then put in a thermal cycler using specific programme for MJ DNA Engine machines (as shown in Table ‎2.1).

Temperature Time Cycles

95C 3 minutes 1X 95C 59C 72C 20 seconds 15 seconds 15 seconds 35X 72C 7 minutes 1X 4C Hold

Table 2.1;The PCR programme used described in terms of temperature (column 1), time of incubation (column 2) and the number of cycles performed (column 3). The programme is specific for MJ DNA Engine PCR machines as stated in the protocol manual.

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PCR Quality Control (agarose gel)

2.4.7.3

Each PCR product was run on a 2% agarose gel, as a part of quality control. A 2% agarose (Gibco® BRL) gel containing 400 ng/ml ethidium bromide was prepared in TAE buffer, (2 M Tris base, 50 mM EDTA (pH 8) and 1 M glacial acetic acid); the same concentration of ethidium bromide was maintained in the TAE running buffer. Three µl of each PCR product mixed with 3µl 2X gel loading buffer (Promega) were loaded per well. Each PCR product was compared against a 1kb bp DNA ladder (Promega) to confirm the size of the amplicon. The gel then was run at 120V for 1 hour.

2.4.8 PCR purification and elution with QIAGEN Min-Elute 96 UF PCR purification plate

All 4 PCR products of each sample (400µl) were consolidated into a single well of a Min- Elute 96 UF PCR Purification Plate. After preparing a vacuum manifold and adjustment to create a vacuum source able to maintain ~800 mbar, the Min-Elute 96 UF PCR Purification Plate was placed on top of the manifold. Unused wells were covered with PCR plate cover and the used well left uncovered. Vacuum applied and the ~800 mbar vacuum maintained until the wells were completely dry. That has taken around 2 hours to dry 400 μl PCR samples.

The PCR products were washed by adding 50 μl molecular biology water and drying the wells completely (approximately 50 minutes). The washing steps were performed 3 times. Then the Min-Elute plate was removed from the vacuum manifold and gently tapped on a stack of clean absorbent paper to remove any liquid that might remain on the bottom of the plate. To elute the PCR product, 40 μl elution buffer (EB) (10mM Tris-HCl, pH 8.5) was added to each well, the plate covered with PCR plate cover film and been moderately shaken on a plate shaker at 960 rpm for 4 minutes. The purified PCR product was recovered by pipetting the eluate out of each well.

2.4.9 Purified‎PCR‎product’s‎concentration‎adjustment

Purified PCR product needed to be at a concentration of 20µg per 45µl solution to be prepared for the next step (Fragmentation). EB (10mM Tris-HCl, pH 8.5) was used for any required dilutions.

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Quantification of purified PCR product

2.4.9.1

Purified PCR products were measured (quantified) by Nano Drop (from Thermo Fisher Scientific Inc), and 20µg per 45µl of each Purified PCR product was prepared for Fragmentation.

2.4.10 Fragmentation

Fragmentation Buffer (5 μl 10X) was added, (using fragmentation plate), to each 45µl of purified PCR products on ice and vortexed at medium speed for 2 seconds. Fragmentation reagent was diluted (0.048 U/µl) on ice, by adding 5µl buffer to 1µl fragmentation reagent and the volume completed to 50µl by molecular biology water. Then 5µl of the mixture was added to the previous 50µl. The fragmentation plate was covered with a plate cover and sealed tightly. The fragmentation plate was vortexed at medium speed for 2 seconds, and briefly spun at 2,000 rpm for 1 minute. The plate was then placed in pre-heated thermal cycler (37 C) immediately for 30 minutes then at 95 C for 15 minutes and then cooled at 4 C.

Fragmentation Quality Control (agarose gel)

2.4.10.1

The fragmented PCR products (4µl) were mixed with 4µl 2X gel loading dye and run on a 4% agarose gel at 120V for 30 minutes (as quality control check), the 100 bp ladder was used as a size standard. After confirming the success of fragmentation reaction by analysing the gel picture, the samples then were handled to a third party, the Central Biotechnology Services (CBS) department, for labelling, hybridization, washing, staining and scanning.

2.4.11 Labelling

Master mix for labelling reaction was prepared (in ice) by mixing 14µl of 5X TdT buffer with 2 μl GeneChip DNA labelling reagent (5 mM) and 3.4 μl of TdT (30 U/μl). A total volume of 19.4 µl of this mixture was added to 50.6 fragmented PCR products making the total reaction of 70µl. (The remaining fragmented DNA was used in the agarose gel). The plate was then sealed tightly with a plate cover, vortexed at medium speed for 2 seconds, and briefly spun at 2,000 rpm for 1 minute. The plate was then placed in pre-heated thermal cycler (37 C) for 2 hours then at 95 C for 15 minutes and cooled down at 4 C. The plate was briefly spun at 2,000 rpm for 1 minute after the labelling reaction.

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2.4.12 Target Hybridization

Hybridization mix was prepared according to manufacturer's instructions. Each labelled sample was transferred from the plate to a 1.5 ml Eppendorf tube and 190μl of the hybridization Cocktail Master aliquoted. The 260 μl of hybridization mix and the labelled DNA were heated at 95 C in a heat block for 10 minutes to denature, and then cooled down on crushed ice for 10 seconds. That was followed by a brief spin at 2,000 rpm for 1 minute in a microfuge to collect any condensate. The tubes were then placed at 48 C for 2 minutes and 80 μl of the denatured hybridization mix injected into the array. It was then hybridized at 48 C for 16 to 18 hours at 60 rpm.

2.4.13 Washing, Staining, and Scanning

Fluidics Station 450/250 automatic washing and staining probe arrays were used according to manufacturer's instructions for GeneChip® mapping. That was followed by scanning of the probe arrays using the GeneChip® Scanner 3000, according to manufacturer's instructions.

2.4.14 Data acquiring

GCOS software (described in ‎2.2.15) was used to acquire the data from the GeneChip® Scanner 3000. The software creates images and cell intensity files, by extracting the measured intensity of raw signal. Thus special files were created for analysis; “Data Transfer Tool (.DTT) Archive files”. These files contain the genotyping data from all members of family #1. Such files are not compatible with Microarray Suite (MAS) and thus GCOS restores these files and made them suitable to be processed by GTYPE software (described in ‎2.2.9). GTYPE has then been used to expose the raw image data of cell fluorescence intensity (.CEL files), which contain information about the expression levels of the individual probes. GTYPE then generates CHP files (from CEL files), for each sample to display a Relative Allele Signal (described in ‎2.2.9) and thus the genotype of each DNA sample across the ~ 10,200 markers (Figure ‎2.15). All data have been saved in Excel format which will be analysed in chapter 3.

2.4.15 Data Check per Individual, per SNPs and per Batches

The data imported into an Excel worksheet has undergone a range of quality control steps. The first step was to check call rate for each SNP across the 19 DNA samples (18 family members and 1 duplicate, as described in the next section), and remove any SNP showing ‘no

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call’ for more than 4 individuals. The second step was to check call rate for each individual across the 10208 SNPs and remove any individuals showing ‘no call’ for more than 10% of SNPs. The third step was to check for any common error in data from the same batch.