To be convenient for the DNA walking approach, these oligonucleotide primers were chosen at the nearest extremity of the walking direction. Note that the t35S pCAMBIA element is the starting position selleck compound and the walking direction
is defined on the rice genome through the left border of the transgenic cassette (Cambia, 2013 and ClustalW2, 2013). Via a different combination, the same oligonucleotide primers were usable for qPCR assays. The oligonucleotide primers and the obtained amplicon sequences are indicated in Table 2 and Fig. 1. The specificity of oligonucleotide primers was initially evaluated in silico using the program “wprimersearch” from the software “wEMBOSS”, which mimics PCR amplification ( Barbau-Piednoir et al., 2012b and wEMBOSS, 2013) ( Table 1). As previously described, for all qPCR assays, a standard 25 μl reaction volume was applied containing 1× SYBR®Green PCR Mastermix (Diagenode, Liège, Belgium), 250 nM of each primer and 5 μl of DNA (10 ng/μl). The
qPCR cycling program consisted of a single cycle of DNA polymerase activation for 10 min at 95 °C followed by IDO inhibitor 40 amplification cycles of 15 s at 95 °C (denaturing step) and 1 min at 60 °C (annealing–extension step). The program for melting curve analysis was performed by gradually increasing the temperature from 60 to 95 °C in 20 min (±0.6°/20 s) (Barbau-Piednoir et al., 2010 and Broeders et al., 2012c). All runs were performed on an iQ™5 real-time PCR detection system (BioRad, Hemel Hempstead, UK) or an ABI 7300 qPCR system (Applied Biosystems, CA, USA) for the specificity assessment and the rest of the analysis, respectively. Concerning the qPCR method acceptance parameters, evaluation of specificity, sensitivity and inter-run repeatability was carried out as previously described (Broeders et al., 2012c). In brief, the specificity of the t35S pCAMBIA c-F and the t35S pCAMBIA a-R primers was tested on several WTs, GMOs and LLPs (Low Level Presence) by qPCR SYBR®Green method using Ct and Tm values as criteria (Tables Table 1 and Table 2) ( Reg. EC no. 619/2011).
Sensitivity and repeatability were determined for t35S pCAMBIA primers on Bt rice using the qPCR SYBR®Green method on Interleukin-3 receptor serial dilutions going from 2000 to 0.1 haploid genome equivalents (HGEs) (Tables Table 2 and Table 4). From these serial dilutions, the PCR efficiency and linearity (R2) were estimated. The t35S pCAMBIA amplicon was cloned into a pUC18 plasmid (INVITROGEN, CA, USA) to obtain the t35S pCAMBIA Sybricon as previously described (Barbau-Piednoir et al., 2010, Broeders et al., 2012c and Sambrook and Russell, 2001). Briefly, the t35S pCAMBIA amplicon was first subcloned into the pCR®2.1-TOPO® Vector using the TOPO TA Cloning® Kit (INVITROGEN, CA, USA) according to the manufacturers’ instructions. After EcoRI restriction, the correct amplicon was then cloned into the vector pUC18 (INVITROGEN, CA, USA).