2.3 Nucleic acid isolation and PCR confirmation
Plant genomic DNA will be extracted from 100mg in vitro plant material i.e. seeds using the CTAB (cetyltrimethylammonium bromide) method using DNA extraction buffer 1% (w/v) SDS, 100mM NaCl, 100mM Tris base, 100mM Na2EDTA, pH=8.5 by HCl), as described earlier by Murray et al., (1980). PCR Primer (PGluB-4 Forward: 5’-gcacgccagaaaatataatgata-3’; TM-1 Reverse: 5’- cagtacgccgagcatact-3’) were utilized to confirm the transgene integration. The Polymerase Chain Reactions (PCR) analysis was conducted for the presence of DNA encoding vaccine antigen. Specific primers to promoter region and TM-1 gene amplified the sequences in transgenic wheat seeds tissues. The DNA extracted from transgenic wheat seeds was used as a template for PCR analysis. The amplification for TM-1 was carried out at 94°C for 5 min, 94°C for 1 min, 61°C for 1.5 min and 72°C for 1 min, with total of 35 cycles, and final extension of 72°C for 10 min. The amplified PCR product was resolved at 1% SDS gel electrophoresis and was finally analyzed under UV light to confirm the presence of estimated band size. After confirmation, all the positive plants were self-pollinated to obtain T1 generation and were utilized for all analysis.