The experiment was also evaluated http://www.selleckchem.com/products/Trichostatin-A.html as repeatable according to the RSDr percentage (relative standard deviation, RSD, of the test results). In addition, the PCR efficiency (80%) and the linearity (R2 = 0.9954) were assessed as acceptable. Following a positive signal observed in qPCR SYBR®Green assay, the second step was to characterise the junction
between the transgenic cassette and the plant genome to confirm the presence of pCAMBIA unauthorised GMOs in food/feed matrices (Fig. 3). Therefore, a DNA walking approach has been developed. In order to supply an integrated approach, an additional oligonucleotide primer, named t35S pCAMBIA b-R, was designed manually, on the conserved region of the t35S pCAMBIA sequence, localised between the t35S pCAMBIA a-R and t35S
pCAMBIA c-R primers previously used for the qPCR SYBR®Green assay (Fig. 1 and Table 2). The specificity of this primer was confirmed in silico via the software wEMBOSS (data not shown). The amplicons resulting from the double semi-nested PCR were visualised on a 1% agarose gel. For each kind of DRT primers mixes Alectinib manufacturer (A–D), amplicons were observed with an approximate size of 300 bp up to 1000 bp (Fig. 2A). The identity of the amplicons was confirmed by direct sequencing of the purified PCR products. The sequencing of the plasmids containing these amplicons allowed identifying the t35S pCAMBIA c-R and UAP-N1/UAP-N2 primers and determining the exact size of the amplicons (408–944 bp) (Fig. 2). All analysed amplicons presented a sequence including
the junction between the pCAMBIA vector and the rice genome. Two transgenic insertions have been detected. For the majority of the amplicons (A2, A3, B1, C2, D1, D2 and D3), Sinomenine the pCAMBIA cassette was integrated on a genomic sequence (OSJNBb0111B07) from the chromosome III of O. sativa japonica Group coding for a putative uncharacterised protein. For the three other amplicons (A1, B2 and C1), the transgene flanking region was localised on a genomic sequence (OSJNBa0016G10) from the chromosome II of O. sativa japonica Group coding for a putative uncharacterised protein. These transgene flanking regions present a shorter left ends compared to the pCAMBIA cassette situated on the chromosome III. This variability of length could be explained by the fact that a left end integrates less precisely than a right end ( Gheysen et al., 1991 and Krizkova and Hrouda, 1998). To confirm the two chromosomal insertions, a PCR amplification using primers annealing to the pCAMBIA construct and the rice chromosome II or III was carried out ( Table 2). The presence of PCR amplification as well as the sequencing of these amplicons allowed verifying properly the transgene flanking regions (data not shown).