Figure 5
From: Cyclic Digestion and Ligation-Mediated PCR Used for Flanking Sequence Walking
Target flanking sequence amplification and T-DNA insertion sites of transgenic Arabidopsis At-Gt75. M: GeneRuler 1-kb DNA ladder. LB: the left border of T-DNA; RB: the right border of T-DNA; OSP1 and OSP2 are specific nested primers (Forward primer) designed in the downstream of OCS terminator, AP1 and Ap2 are specific nested primers (Reverse primer) designed in adapter, GJ4-F and DJ-R are detection primers designed according to the right flanking sequence and the vector right boundary sequence, and GJ4-F and DJ-R are detection primers designed according to the right flanking sequence and the vector right boundary sequence; all the primers sequence used here are listed in Table 2. (a) The second round of PCR products amplified from transgenic Arabidopsis At-GT75 with primers OSP2 and AP2, 2 bands recovered from lane 1 and lane 3 are sized 1067 bp and 1411 bp by sequencing; (b) The right boundary PCR detection, lane 5 is a 2.3 kb PCR band with primers GJ4-F and GJ-R and lane 6 is 1.2 kb PCR band with primers GJ1-F and GJ-R. (c) Sequencing results from band 1and 5 showed that one of the T-DNA insertion sites in At-Gt75 was between 7375617 bp and 7375618 bp on Chromosome 4 of the Arabidopsis genome; (d) Sequencing results from band 2 and 6 showed that another T-DNA insertion site in At-Gt75 was between 27678316 bp and 27678317 bp on Chromosome 1 of the Arabidopsis genome.
