Molecular Genetic Techniques

The Polymerase Chain Reaction (PCR) was made use of to amplify the DNA. The GoTaq® Flexi DNA Polymerase kit or Herculase II Fusion DNA polymerase were employed. The total PCR reaction volume per 50 µl, contained the components in Table 2- 1.

Table 2- 1: PCR Reaction Reagents per 50 µl PCR Reaction

PCR Reagent Volume (µl)

Water 32.75

5x Buffer 10

25 mM MgCl 3

10 mM dNTPs 1

Taq polymerase 0.25 (units)

Forward and reverse Primers

(20µM each)* 2

DNA 3 (1 to 100 ng)

*Details of primers are discussed separately.

PCR was performed in an Eppendorf Mastercycler 5333 (AG Eppendorf, Hamburg, Germany) using the optimised programme as follows:

94°C for 3 minutes (Initial denaturation)

94°C for 30 seconds (Denaturation)

60 or 65°C for 30 seconds (Annealing)

92 Repeat steps 2-4 for 32 cycles using (Go Taq) or 10 cycles using (Herculase II) polymerase reagents

72°C for 5 minutes (Final extension)

END

2.7.2. PCR Primer Design

Primers were designed according to Oligo Analyzer Version 3.1 (Integrated DNA Technologies (IDT, Surrey, UK).

2.7.3. Sanger Fluorescent Dideoxynucleotide Sequencing

PCR products were custom sequenced by separately using their corresponding forward and reverse PCR primers (Genewiz® Sanger Service, Takeley, UK).

2.7.4. DNA Fragmentation

DNA was mixed with Ion Shear Plus 2.5 µl of 10x reaction buffer and nuclease-free water to 25 µl. Then, 1 µl of Ion Shear Plus enzyme mix was added. The reaction mixture was incubated in a water bath at 37°C for 18 minutes. Five μl of Ion Shear Plus stop buffer was added and the sample was stored on ice.

2.7.5. DNA Adapter Ligation

A 75 μl reaction master mix was prepared per sample containing: 7.5 μl 10x ligase buffer; 7.5 μl Ion Xpress P1 adapter; 2 μl dNTP mix; 31 μl nuclease-free water; 4 μl DNA ligase; and 8 μl nick repair polymerase. 10 μl of one of the barcode adaptors (KAPA Biosystems,

93 Boston, USA) was added per sample. The ligation reaction was then incubated in a

thermal cycler at 25°C for 15 minutes followed by 72°C for 5 minutes and held at 4°C.

2.7.6. DNA Capture Hybridisation

750 ng of genomic DNA per 3.4 μl of sample was required. Samples with a DNA

concentration of < 221 ng/μl were therefore concentrated in a vacuum concentrator at ≤ 45°C. Regarding SureSelect hybridization buffer, 49 μl per sample was prepared by mixing 25 μl Hyb#1, 1 μl Hyb#2, 10 μl Hyb#3 and 13 μl Hyb#4, 40 μl was used. 2 μl of the

SureSelect capture library was added to 5 μl of 10% RNase block per sample. Additionally, 5.6 μl of SureSelect block mix was prepared per sample using 2.5 μl Block#1, 2.5 μl

Block#2, and 0.6 μl Block# 3. 3.4 μl of the 221 ng/μl each sample was added to a 0.5 μl PCR tube and 5.6 μl of SureSelect Block mix added before heating at 95°C for 5 minutes and lower the temperature to 65°C in a thermal cycler with the lid set to 105°C. 40 μl of hybridization buffer was added to a 0.5 μl PCR tube per sample and transferred to the thermal cycler maintained at 65°C, at which point the tube was incubated for 5 minutes. Seven μl of SureSelect capture library mix per sample was added to a 0.5 μl PCR tube and placed into the thermal cycler at 65°C for 2 minutes. The tubes were maintained in the thermal cycler and each prepped sample was transferred to a SureSelect Capture Library tube, with the contents then being mixed by pipetting 10 times and tube capped in the thermal cycler to allow hybridisation to proceed for 24 hours.

2.7.6.1. Magnetic Bead Preparation

SureSelect Wash 2 was prewarmed at 65°C in a water bath. 50 μl of resuspended Dynabeads MyOne Streptavidin T1 beads (Thermofisher Scientific, Loughborough, UK)

94 were added to a 1.5 ml LoBind tube and vortexed. The tube was then placed into the Dynal magnetic separator (Thermofisher Scientific, Loughborough, UK) and the

supernatant removed. This latter step was repeated twice, each time re-suspending the recovered beads in 200 μl of SureSelect binding buffer.

2.7.6.2. Hybrid Capture

The hybridized sample was directly added to the bead solution, and the tube was then inverted three to five times to mix and incubate for 30 minutes at room temperature. A magnetic separator was used to separate the beads and their captured DNA from the buffer and the supernatant with uncaptured DNA was removed. Beads were washed by adding 500 μl of SureSelect Wash 1 and mixed by vortexing before incubation for 15 minutes at room temperature with occasional mixing by vortex. Beads were recovered as before and 500 μl of the pre-warmed SureSelect Wash 2 was added, mixed by vortexing and incubated for 10 minutes at 65°C with occasional mixing by inversion for further washing. Recovery of the beads was performed as before, and a second washing step was repeated for a total of three times. Lastly, 30 μl of nuclease-free water was added to the beads, with the targeted DNA enrichment retained.

Target enrichment was performed by cRNA hybrid capture according to the SureSelect TE System (Agilent technologies, Shrewsbury, UK). This included cRNA probes according to SureSelect protocol, which captures regions totalling from 0.5 Mb up to 2.9 Mb. The steps required involve selection of cDNA probes, DNA sample preparation for hybridisation, hybridisation capture and post-hybridisation amplification (256). Selection of

95 appropriately sized fragments was performed at intermediate stages. Figure 2.1 outlines the relationships between the steps with the Sure Select TE System.

Figure 2- 1: The Steps of SureSelect Target Enrichment System (Agilent). Additional details are found in the manufacturer’s protocol; SureSelect Target Enrichment System for Sequencing on Ion Proton (257).