Figure 3

One-step assembly of the functional gene in eukaryotic in vitro translation system. The mutated DNA, the diagnostic complex and the non-assembled gene components were mixed in equimolar ratio and added to the in vitro translation reaction (lane: one step self-assembly). The translation product with a molecular mass of 17.3 kDa is marked by asterisk. Reaction containing only the intact hID1 gene served as positive control (lane: positive control); reaction lacking the mutated DNA was used to test the self-ligation of the diagnostic complex (lane: self-ligation control). The low concentration of the output product severely restricted the detection techniques that can be used to detect the translation product, i.e., in this case only by radioactivity. Note that the eukaryotic transcription/translation coupled system gives two high background signals at 10–13 and 28 kDa. The background that arises from the in vitro translation kit itself was tested in a negative control reaction containing pIVEX vector without any insert (lane: negative control). P and S denote pellet and supernatant, respectively.