Polymerase Chain Reaction

2.2.6.1 Conventional PCR

Exons that required mutation screening were amplified using the polymerase chain reaction (PCR) (Mullis et al. 1986). Standard 25 μl PCR reactions were used. Each

reaction contained 25 ng genomic DNA, 100 ng of each specific primer (forward and reverse), 1 μl deionised water and 22.5 μl Reddymix® (1.25 U Taq polymerase, 75 mM Tris-HCl, 20mM (NH4)2SO4, 0.01% (w/v) Tween20, 0.2 mM of each dNTP and 1.5 mM MgCl2 (Fisher Scientific UK Ltd.). A conventional PCR includes cycles of 3 essential steps: denaturation, annealing and extension (Figure 2.1). The cycling conditions used in this study were; initial denaturation step at 94ºC for 7 min, followed by 35 cycles of denaturation at 94ºC for 1 min, annealing at nºC for 1 min and extension at 72ºC for 1 min, and a final extension step at 72 ºC for 7 min (Figure

2.1). The annealing temperatures varied according to the TM of the primers (Appendix 1). Throughout the project, all PCR reactions were carried out using the GeneAmp® PCR System 9200 thermocycler (Applied Biosystems).

35 cycles of: denaturation (at 94ºC), annealing (depends on primers TMºC), extension (at

72ºC)

2.2.6.2 Long-range PCR

Long-range PCR is used to amplify larger fragments of DNA than those achieved using conventional PCR protocols (Cheng et al. 1994). Modifications from the conventional PCR protocol included the use of thermostable DNA polymerases with high 5‟-3‟ polymerase activity and 3‟-5‟ proofreading capability. Additionally, the

extension step in the long-range PCR protocol was usually longer than in conventional protocols to allow the amplification of large fragments. Long-range PCR was performed using the Phusion high fidelity PCR kit (New England Biolabs (UK) Ltd., Hitchin). Long-range PCR reactions were set up in a total volume of 50 μl and consisted of 62.5 ng genomic DNA, 0.5 μM of each primer (forward and reverse), 32.5 μl deionised water and 10 μl of 5x Phusion HF buffer®, 200 μM of each dNTP

and 10 ul of the Phusion DNA polymerase. The long-range PCR conditions consisted of the following steps; initial denaturation at 98ºC for 30 sec, followed by 35 cycles of denaturation at 98 ºC for 10 sec, annealing at nºC for 30 sec and extension at 72 ºC

7min 1min 1min 1min 7min 14 C 94 C

n C

72 C

35 cycles 10min

for x min, and a final extension at 72ºC for 10 min. The annealing temperatures varied according to the TM of the primers (Appendix 1) and the extension time varied according to the size of the target region (15-30 sec/Kb). The PCR conditions were determined according to the Phusion kit recommendations.

2.2.6.3 GAP PCR

GAP PCR is a modified PCR that allows the detection of known insertions and deletions. It involves the use of 3 primers: a “common” forward primer, which anneals

to the sequence upstream of the insertion/deletion site and 2 reverse primers. The first primer “insertion” anneals to the inserted sequence when present, and the 2nd primer “deletion” will anneal downstream of the insertion/deletion sequence (Figure 2.2). In the case of a homozygous deletion, the “common” and the “deletion” primers alone will produce a product as the “insertion” primer will fail to anneal. In the case of an insertion, the “common” and the “insertion” primers alone will produce a PCR

product as the optimised PCR conditions do not allow the amplification of large PCR products. The genotype of the sample is determined by gel electrophoresis based on the size of the different products (Figure 2.2). In this project GAP PCR was initially used for genotyping the CNV23598. Standard conventional PCR reactions and conditions were used, however, with 3 primers (Section 2.2.6.1).

2.2.6.4 Fluorescent GAP PCR

Fluorescent GAP PCR was used as a higher throughput method for the genotyping of CASP8 CNV23598. The principle is the same as described in the previous section. However, the “insertion” and the “deletion” primers were labelled with distinct fluorescent dyes and instead of gel based analysis, capillary electrophoresis was used. The products appear as peaks of different size and fluorescent colour according to the different alleles. Fluorescent GAP PCR was performed using the Amplitaq Gold® PCR master mix (Applied Biosystems). The reactions were set up in a 10 μl volume and consisted of 5 ng DNA, 0.5 μM of the unlabelled common forward

primer, 0.5 μM of the “insertion” labelled reverse primer, 0.2 μM of the “deletion” labelled reverse primer, 5 μl of the Amplitaq Gold® PCR master mix and 2.8 μl deionised water. The PCR conditions consisted of the following steps; initial denaturation at 95ºC for 5 min, followed by 32 cycles of denaturation at 95 ºC for 30 sec, annealing at 59ºC for 30 sec and extension at 72 ºC for 30 sec, and a final extension at 72ºC for 7 min. The products were diluted 1 in 10 with deionised water and then electrophoresed on ABI PRISMTM 3730 DNA analyser with appropriate size standard by the Core Sequencing Facility (School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield, UK). The data were analysed using genemapper software version 4.0 (Applied Biosystems).

A schematic presentation of GAP PCR. Arrows represent the primers used. Black is the common forward, Red is the insertion reverse and Blue is the deletion reverse A) The target sequence with an insertion, the reverse deletion will anneal, however, the PCR conditions will not allow the amplification of large amplicons. B) The target sequence with the deletion, the insertion reverse will not anneal C) The different genotypes as will appear on agarose gel.

Homo INS Heterozygous Homo DEL

Target Insertion Target A B C Figure 2.2 GAP PCR