Polymerase chain reaction (PCR)

The PCR is a technique that amplifies DNA by using the enzyme DNA polymerase. Two pre-designed oligonucleotides (primers) anneal to distinct, complementary parts of the DNA, one at the 5’-position, the other one at the 3’- position of the desired fragment to be amplified. Then, the polymerase, traditionally derived from thermus aquaticus, hence called Taq polymerase, extends the DNA downstream (5’!3’) from the primer, generating a new DNA strand. This strand serves as a template for the generation of further DNA strands in subsequent cycles of the reaction, yielding an exponential amplification of DNA fragments.

The standard Taq polymerase was purchased from Bioline (London, UK); for PCR-based cloning, a mixture of two polymerases, Taq- and Tgo-polymerase, were used, as the latter one, derived from thermococcus gorgonarius, possesses a pronounced 3’!5’ exonuclease activity. This ‘proofreading’ ability of the enzyme reduces the error rate from 1.3 x 105 (Taq) to 4.9 x 107 (Tgo) resulting in a high fidelity of DNA synthesis (Keohavong and Thilly, 1989). The enzymes were

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___________________________________________________________________ purchased from Roche as part of the Expand High Fidelity PCR System™ (Roche, Basel, Switzerland).

All reactions were carried out in a 25 µl total reaction volume. The following components were added (final concentration): reaction buffer (1x), MgCl2 (1.5-4 mM),

forward and reverse primer (0.4 µM), dNTPs (200 µM each) (Bioline, London, UK), DNA polymerase (0.05 U/µl), 10-15 ng of DNA template.

Reactions were carried out in 200 µl reaction tubes (Sigma, Pool, UK) in a thermal cycler (TProfessional Basic gradient, Biometra, Goettingen, Germany). After one initial denaturation step (95°C, 5 minutes), 32-35 cycles followed with 1) 95°C for 1 minute (denaturation), 2) 60°C for 30 seconds (primer annealing) and 3) 72°C between 30 seconds and 2 minutes (elongation). After the last cycle, a final elongation step of 5 minutes at 72°C was performed. The duration of cycle step 3) depends on the length of the DNA fragment to be amplified; it was calculated based on the capacity of the polymerase, which is about 1000 bp per minute. Fragments were analysed by agarose gel electrophoresis (2.2.4).

2.2.10. Real-time quantitative (RTQ) PCR

RTQ-PCR is an enhancement of the PCR and measures the amplification of DNA strands at each cycle, i.e. in real time. Essentially, it has the same components of a standard PCR; its principle relies on the 5’!3’-exonuclease activity of the polymerase and the presence of a 20-60 bp oligonucleotide probe that binds to the DNA template in-between the primer pairs. This probe has a fluorophore at its 5’ end and a quencher at its 3’ end, that silences the emission of fluorescence when both quencher and probe are in close proximity, as in intact oligonucleotides. During amplification, the probe binds to the synthesized DNA strands and gets degraded by the 5’!3’-exonuclease activity of the enzyme, which removes the quencher from the

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___________________________________________________________________ fluorophore. Then, the probe is able to emit fluorescent light when excited and the amount of fluorescence detected in the sample directly correlates with the amplified DNA in the sample, but it also correlates to the amount of DNA template that was initially added into the PCR reaction.

During the initial RTQ-PCR cycles, the DNA is amplified exponentially, until the amplification is saturated, mainly as a consequence of the consumption of the dNTPs or exhaustion of the enzyme. To compare the amount of DNA in a sample, a threshold needs to be determined when the DNA is amplified exponentially; the threshold allows the calculation of threshold cycles (Ct) for each target, which essentially correspond to the amount of template added to the sample or the mRNA- expression in a sample, whose RNA was extracted and transcribed into cDNA by RT reaction. Importantly, the lower the Ct values are, the higher the gene of interest is expressed. To determine the relative expression of a gene, the Ct values of a constantly expressed housekeeping gene (i.e. 18S) can be subtracted from the gene- specific Ct values; these resulting delta Ct (dCt) values correct for equal sample loading and were used for calculations.

All assays (a pre-mix of gene-specific primers and probes) and the Taq polymerase mastermix were purchased from Applied Biosystems (Quarrington, UK). The RTQ-PCR reactions were carried out in a 7500 ABI qPCR machine (Applied Biosystems, Quarrington, UK) and the data were analysed with the product-specific software. The total reaction volume was 10 µl, consisting of 5 µl mastermix, 0.5 µl TaqMan gene expression assays and 4.5 µl of cDNA sample (see 2.2.3; 7.8 ng mRNA-equivalent cDNA). Reactions were carried out in duplicate and 18S was used as a housekeeping gene. All TaqMan assay probes were labelled with 6- carboxyfluorescein (FAM), the 18S probe with the VIC® _fluorophore.

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2.2.11. Sequencing analysis

DNA sequencing analysis was performed after obtaining informed consent and assent from patients and their parents with approval of the local institutional review board committees.

After genomic DNA extraction from peripheral leukocytes utilizing a blood and cell culture DNA kit (Qiagen, Hilden, Germany), the mutation analysis was performed by PCR of the respective gene with primer pairs covering the whole coding region, including the intron-exon boundaries (Table 7). The PCR conditions are described in section 2.2.9. The PCR fragments were checked for integrity and correct size (Table 7) by agarose gel electrophoresis (section 2.2.4) and subsequently purified with the QIAquick PCR purification kit, following the manufacturer’s protocol (Qiagen, Hilden, Germany). 5-10 ng of purified DNA template with 3.2 pmol of primer (the same as used for PCR amplification; see Table 7) was processed for electrophoresis on an automated ABI PRISM 310 Sequencer and analysed with the ABI SeqScape 1.1 software (Applied Biosystems Inc., Weiterstadt, Germany). Sequencing analysis was performed using Lasergene® software (DNASTAR Inc., Madison, USA) and mutation numbering was carried out referring to the NCBI reference sequences: POR: NG_008930.1 (genomic) and NP_000932 (protein); CYP17A1: NG_007955.1 (genomic) and NP_000093 (protein); CYB5A: NG_023211.1 (genomic), NP_683725.1 (protein). For DNA numbering, the nucleotide designated +1 was the A of the ATG start codon.

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Table 7: Primer sequences, annealing temperatures (Tm) and expected fragment sizes following PCR amplification of the exons of CYP17A1, POR and CYB5A from genomic DNA.

Name Sequence (5’-3’) Tm (C) Fragment size (bp) CYP17A1 CY17A1 - 1F TCTAGGCTCAGAGAGAGGTG 65.1 650 CY17A1 - 1R GGGCTCCAGGAGAATCTTTC 63.9 CY17A1 - 2F GGTGTGAGATTCCTACGCC 61.1 676 CY17A1 - 3R TCTACAGAACCTGAAGGCAG 57.8 CY17A1 - 4F GGTGGAGTAGGAACTTCCAS 60.1 316 CY17A1 - 4R TGTGCCAGGTTCTCTGCTT 66.6 CY17A1 - 5F CCTGCCCAGACTTGCTCTAC 63.9 935 CY17A1 - 6R CTGACTTTAGGTTGGCCAGCA 66.3 CY17A1 - 7F AGCTGTTCAGACAGAAGCGC 64.6 1259 CY17A1 - 8R GGCATTGCCACAAGCTGAAA 68.3 POR

POR - 1F AGT GAC CAT TTC CTG CAG 58.7 368

POR - 1R ACT GCT TGG AGT GGT GAC AG 63.0

POR - 2F ATG ACA CCT GCC TCC CAC 63.8 159

POR - 2R GAC TTG ATT ACA TGC CCA TCG 64.1

POR - 3F GTG GCT GAG GTC TGT GGC 64.9 241

POR - 3R TAC AGA CCT GCT CCC TGT CC 64.3

POR - 4F TTG AAC AGG CTC AGT CAT GG 63.9 236

POR - 4R TGG AGT CCC CAG GGA GG 66.5

POR - 5F CCA CTG GTC AGG TCG AGG 64.6 232

POR - 5R CAC GGC CCC TGC CTA AG 66.8

POR - 6F ACA GTC CTG AGC TTT GGG G 64.2 303

POR - 6R CTT CTA ACC TTG CTG CGA CC 63.7

POR - 7F TGT AGT CCA ACC CCT CCC TC 65.2 218

POR - 7R AGT GGC CAT AGA GCC GTC TG 66.6

POR - 8F GTG CTT TGT GCA ACC AGA AG 63.5 515

POR - 9R GCC TAA GCA GAA GCT CAA CC 63.0

POR - 10F GAG CAT AGG CCT TGT TTC CA 64.0 544

POR - 11R CTT GCA CTC TGC CTG CTG T 64.6

POR - 12F CTG CAG AAC GGG ACT TGG 64.6 649

POR - 13R AAG GGT GGT GCT GTG AGG 63.9

POR - 14F AGC AGT CCC ACA AGG TGA GA 64.9 583

POR - 15R GGC TGA GGA GGA TGC AC 62.1