Figure 1. Identification of an individual GM cotton seedling using PCR and Southern hybridization.
(a) Identification of an individual GM cotton seedling. PCR was performed in each of the 6 plants (lanes 1-6) of a group tested positive for presence of the CaMV 35S promoter. Lane 1 shows the band from the sample containing the 35S sequence (195bp) similar to the positive control (P). MW is molecular weight DNA marker and N is negative (no template) control.
(b) PCR products using the primers 35Spro3 and AP1 on the libraries constructed with the Genome Walker kit (BD Biosciences, Palo Alto, USA) from DNA of the GM seedling using the restriction enzymes DraI, EcoRV, PvuII and StuI (lanes D, E, P, S respectively). No clear bands could be detected from the four libraries and the gel was transferred and hybridized with a CaMV 35S promoter -specific probe. C₁ and C₂ are positive PCR controls using GM cotton genomic DNA as template and the primers 35Spro5/35Spro3 and 35Spro5/NOS3 respectively and N is negative (no template) control. MW is the λHind3-φx174 molecular weight DNA marker.
(c) Southern hybridisation of a membrane produced after transfer of the PCR products shown on the gel of Figure 1b with the CaMV 35S promoter -specific probe. Lanes are as in Figure 1b. Two bands were detected corresponding to the libraries D and E. Positive control C₂ gave a strong hybridization signal and C₁ was overexposed and produced a blurred diffused band.