Figure 2.

Detection of ZFN-induced mutations in soybean hairy-root tissue. A, A schematic strategy highlighting the restriction endonuclease PCR assays used to enrich mutated DNA sequences from soybean hairy-root tissue. Five ZFNs were designed to target seven genes, two of which targeted duplicate copies of DCL1 and DCL4 (DCL1a and DCL1b were successfully targeted by one ZFN construct, but are shown in different sections because they were screened with paralog-specific primers; DCL4a and DCL4b were successfully targeted by one ZFN construct and are shown together because they were screened with a shared primer set). A single ZFN capable of targeting both copies of RDR6 could not be identified. The closest match for both RDR6a and RDR6b was a target site that differed by 2 bp. Two different ZFN constructs were designed to target these respective sites. A ZFN was designed to target one of the duplicate copies of HEN1. B, The PCR products from four root samples, including an undigested (WT/U) and digested (WT/D) wild-type control, were separated on a 2% agarose gel. Lanes showing undigested products indicate a portion of the hairy-root cells having novel mutations at the restriction sites.