Figure 1.

Principal steps of the multi-copy bead display approach. The bead display approach involves two steps of compartmentalization using water-in-oil emulsions. In the first step, single template molecules of a DNA library are amplified in each picolitre reactor of an emulsion PCR (emPCR) with a forward primer coupled to microbeads and a reverse primer covalently coupled with BG moieties (1–3). As a result, many copies of the same DNA, each carrying a BG substrate, are immobilized on the bead. These beads are then recovered from the water-in-oil emulsion (4) and are used in a second step in an IVTT reaction also performed in emulsion. The amplicons contain a phage promoter (e.g. T7) upstream of an open reading frame encoding a fusion protein between the protein under selection and the modified DNA repair protein SNAP that reacts specifically with BG moieties incorporated into the amplicons. During the transcription–translation reaction, the fusion proteins synthesized in each picolitre reactor of the emulsion (5,6) are covalently coupled to the BG moieties of the beads present in the same picolitre reactor (7). The beads can then be isolated from the emulsion (8) and can be used in subsequent screening assays.