IPCR strategy for obtaining T-DNA flanking sequences.

<p>The diagram illustrates the IPCR strategy used to obtain sequences flanking the T-DNA insertion sites (ISs). Restriction enzymes (shown as scissors) with recognition sites (black lines) near the T-DNA border sequences (LB and RB) were used to digest DNA from T-DNA insertion lines. Enzymes cut both within the T-DNA and genomic sequence, and ligations were performed to circularize purified digestion products. PCR of the ligation products was performed using T-DNA specific primers and sequencing was performed with nested primers. The orientations and locations of primers with respect to restriction sites are shown as black arrows. Primers located within the T-DNA directed toward the junction with genomic DNA are designated T primers (LB-T and RB-T). Sequencing reactions using these primers return genomic sequence directly adjacent to the IS (dark blue dotted arrows). Primers directed into the T-DNA are designated RE primers (LB-RE and RB-RE). After enzyme digestion and ligation, sequencing reactions using these primers return genomic sequence starting from the closest restriction enzyme recognition site within the <i>Brachypodium</i> genomic sequence and directed toward the T-DNA insertion (light blue dotted arrows).