Standard PCR

A standard PCR contained 5 ng genomic DNA, 0.5 U Taq polymerase (Invitrogen), and final concentrations of 1 x Taq polymerase buffer (Invitrogen), 50 µM each dNTP, and 0.2 mM each primer in a volume of 25 µl. The PCR cycling conditions were 94ºC for 2 min followed by 30-35 cycles of 94ºC for 30 s, annealing temperature according to primer for 30 s, 72ºC for 1 min/kb of the expected product, followed by a final step of 72ºC for 4 min.

Proof reading Taq polymerase was used in the PCR’s to construct vector pR241 and pR261. The PCR reactions (50 µl) contained 1 unit KOD HotStart DNA polymerase (Novagen, Toyobo), 1 x PCR buffer provided by the manufacturer, 1.5 mM MgSO4, 0.4 mM of each primer, and 50 µM of each dNTP. Thermocycle conditions were as described above, but 1U recombinant Taq polymerase was added after the final step of 72ºC for 4 min and incubated for 2 min at 72ºC in order to create A-overhangs for subsequent ligations.

2.10.3

Colony PCR

For screening of E. coli transformants, colonies were picked-up from selective plates and resuspended in a 25 µl PCR reaction and the PCR cycling conditions were as for the standard PCRs but the first step of 94ºC was extended to 4 min.

2.10.4

Real time PCR analyses

Analysis of gene transcription was assessed by relative quantitative RT-PCR using the Faststart DNA MasterPLUS SYBR Green I system (Roche Applied Science). Real time PCR reactions were carried out using a LightCycler® 2.0 and software version 3.5.

Each reaction was performed in a 10 µl volume, containing 2 µl template and 8 µl proprietary PCR mix. The PCR mixed contained 2 µl Faststart DNA MasterPLUS SYBR Green I system and 500 nM each reverse and forward primers in PCR grade water.

Where possible primers were designed to connect two exons (Table 2.2) to avoid any amplification of possible contaminating gDNA.

The PCR program used for all reactions had an initial step of 95°C for 10 min to activate the Faststart Taq DNA Polymerase. Reactions were subsequently subjected to 45 cycles of PCR (10 s of 95°C, 10 s of 59°C, 16 s of 72°C and 5 s at 84°C). The amplified products were detected every cycle during the 84°C step to negate effects of primer dimer accumulation on fluorescence levels.

A melting curve program followed the PCR as follows: the PCR products were heated to 95°C then immediately decreased to 65°C for 15 s and then gradually increased up to 95°C at a rate of 0.2°C/s with constitutive fluorescence acquisition.

2.10.4.1

Creation of standard curves and coefficient files

For real-time RT-PCR analyses a coefficiency corrected calibrator-normalized relative quantification analysis was used as described in the Roche Applied Science Technical Note No. LC 13 (Sagner & Goldstein 2001). Therefore standard curves for each gene of interested were generated. The gene specific cDNA was synthesized as described in Section 2.10.6 using primer pairs which were used in the successive real time PCR reaction for each gene. The PCR product was purified as described in Section 2.3.3 and diluted in a tenfold dilution series over 5 orders of magnitude. Dilutions of the cDNA were used in the light cycler reaction to generate standard curves for each gene and primer pair, with triplicate reactions for each dilution. The data generated by the real time PCR were saved and processed in the RelQuant 1.1.1 software (Roche), according to the manual, to generate coefficient files.

The standard curves were used to create fit coefficients using the RelQuant 1.1.1 software. The software compares the efficiency of the PCR reaction of the target (gene of interest) and reference (control gene, which is constant for all samples) at different

expression analyses to correct for differences in the efficiency of the target and reference reaction. The only classical housekeeping gene known of D. septosporum is the β–tubulin gene tub1. Initial trials suggested that the expression of tub1 is not constant as needed for a reference gene. Therefore the 18S rDNA was used as an endogenous reference gene in this study. Although the concentration of 18S rRNA is higher than that of mRNA in the cells it appeared to be an appropriate reference gene in A. parasiticus (Chang et al. 2004). As the gene expression studies were performed with pure cultures of D. septosporum, it was decided to use the universal fungal rRNA primers NS7 and NS8 (White et al. 1990). All real time PCR products of target genes were designed to have a similar amplification size as the NS7/NS8 product (~400bp). Primers were designed using the Probe Design Software 2.0 (Roche). Example data are shown in Appendix A5.

2.10.4.2

Gene expression analyses

Expression of the dothistromin genes relative to the 18S rDNA was determined in duplicate for each of three biological replicates. Expression of tub1 was included in the expression experiments to show that the calculated expression levels did not result from variations in the level of 18S rDNA. Samples were analysed in triplicate from a 10-fold or 100-fold dilution of the original cDNA. Two technical replicates of three independent samples (biological replicates) were used for calibrator-normalized relative quantification analysis. Levels of gene expression in each sample were calculated against the endogenous 18S rRNA gene, as a reference.

In each light cycler run a calibrator for the 18S rDNA and for the target gene were included in duplicate. The calibrators for target and reference gene had the same concentration for each reaction of one experiment. The differences between the target and reference calibrators were used to normalize each reaction by correcting for differences between reactions (as shown below).