e122 Nucleic Acids Research, 2009, Vol. 37, No. 18 PAGE 6 OF 9 Figure 5. (A) The enrichment of Avi-AGT DNA in model aﬃnity selections relative to a non-binding, control template coding for AGT-FRB was >250-fold. L1, hyperladder I in kilobase pairs; L2, PCR ampliﬁcation of the supernatant; L3, PCR ampliﬁcation of the bead fraction. (B) Assembly of recovered DNA fragments; L4, hyperladder I in kilobase pairs; L5, substrate AGT recovered from the bead fraction with PfuTurbo Cx, precipitated with PEG–MgCl2 and blunted with T4 DNA polymerase; L6, assembly of L5 with its UTRs + Avi-tag. L7: purifying PCR of L6 with 50 ng DNA per 100 ml PCR; L8, hyperladder I in kilobase pairs; L9, substrate AGT recovered from the bead fraction with PfuTurbo Cx and blunted with T4 DNA polymerase; L10, assembly of L9 with its UTRs + Avi-tag; L11, purifying PCR of L10 with 50 ng DNA per 100 ml PCR. Every sample lane contains 300 ng DNA. selections (Figure 5). For this purpose, BG labelled DNA templates coding for Avi-AGT and AGT-FRB (FKBP12-rapamycin-binding domain) were mixed at a ratio of 1:250 and then compartmentalized along with an in vitro TS–TL reaction in a water-in-oil emulsion (13,27). Following expression and conjugation, the result- ing protein–DNA conjugates were then extracted from the emulsion and the templates coding for Avi-AGT enriched on streptavidin-coated paramagnetic microbeads. This was followed by recovering the DNA in the supernatant and microbead fraction by PCR using primers that annealed to both templates (Figure 5, L2-3). DNA coding for Avi-AGT could only be detected in the microbead fraction (Figure 5, L3) and not in the super- natant (Figure 5, L2); this conﬁrmed the ﬁdelity of the selection process along with a >250-fold enrichment of Avi-AGT over AGT-FRB. In parallel, only the gene coding for AGT was recovered from the microbead frac- tion using primers that were speciﬁc to AGT. PCR pro- ducts were obtained with both types of polymerases, PfuTurbo Cx and Taq (Figure 5, L5, Supplementary Figure S3, L2), and could subsequently be regenerated into their full-length DNA templates (Figure 5, L6-7, Supplementary Figure S3, L3-4). Once again, precipita- tion of the assembly substrate with PEG–MgCl2 proved necessary and suﬃcient for the speciﬁcity of the assembly process and subsequent purifying PCR (Figure 5, L9-11 and Supplementary Figure S3, L6-8). On this occasion, we also noticed that the protein–DNA conjugates did not have to be eluted from the microbeads as PCR ampliﬁca- tion with PfuTurbo Cx remained highly functional in the presence of relatively high concentrations of microbeads ( 6.7 107 M-270 streptavidin coated paramagnetic microbeads per 1 ml PCR). This is surprising considering that paramagnetic microbeads are generally thought to interfere with the performance of PCR. Similarly, we found that the eﬃciency of PCR ampliﬁcation with Taq DNA polymerase can be signiﬁcantly improved by includ- ing b-mercaptoethanol at a ﬁnal concentration of 20 mM in the ampliﬁcation reaction. DISCUSSION A procedure was developed that allows the assembly of a GOI into a linear DNA template with all the components necessary for in vitro TS–TL. Assembly is achieved using a coupled uracil excision–ligation strategy based on USER enzyme and T4 DNA ligase, which allows the simultane- ous and seamless assembly of three diﬀerent PCR pro- ducts with a minimal number of work-up steps by that conferring simplicity, robustness and speed to the method. For its success, several factors proved critical. To achieve a high eﬃciency of assembly and eﬃcient PCR puriﬁcation, it was critical to purify all substrates by PEG–MgCl2 precipitation prior to assembly (24). Otherwise, primer dimers and other small non-speciﬁc by-products that could not readily be visualized with ethi- dium bromide interfered with the assembly process (Figure 2, L8-10). Precipitation with PEG–MgCl2 consti- tutes an inexpensive and fast method to purify PCR pro- ducts. Size dependence can easily be exerted by titrating the optimal concentration of PEG-8000 for a given DNA fragment even though its resolution is less stringent compared to gel puriﬁcation. Nonetheless, precipitation with PEG–MgCl2 proved necessary and suﬃcient in this case to generate clean substrates for eﬃcient assembly through uracil excision–ligation. Despite its widespread applicability to any assembly procedure that features PCR products including over- lap extension PCR and restriction–digestion–ligation,