PAGE 5 OF 9 Nucleic Acids Research, 2009, Vol. 37, No. 18 e122 Figure 3. In vitro expression tests measuring the activity of Avi-AGT following active site dependent labelling of AGT with BG-biotin. DNA templates were either ampliﬁed from assembly reactions or plasmid DNA. L1, no template control; L2, biotinylated protein marker; L3, ampliﬁed with Taq from plasmid DNA; L4, ampliﬁed with Taq from an assembly reaction; L5, ampliﬁed with PfuTurbo Cx from plasmid DNA; L6, ampliﬁed with PfuTurbo Cx from an assembly reaction. The band at 20 kDa corresponds to biotin carboxyl carrier protein. Linker proteolysis can be excluded as a cause for the two bands since biotinylation of the Avi-tag with biotin ligase similarly generated two distinct bands (Supplementary Data and Figure S2). assembly reactions (Figure 2, L3 and Supplementary Figure S1, L3) were ampliﬁed in parallel with an equal amount of a full-length template that served as a compar- ative calibrator. The assembly eﬃciency was determined as 27 and 22% for substrates prepared with Taq and PfuTurbo Cx respectively. This is in good agreement with the assembly eﬃciency of 25–33% estimated from ethidium bromide stained agarose gels (Figure 2, L3 and Supplementary Figure S1, L3). Subjecting the entire assembly mix to repair with the PreCR kit prior to PCR ampliﬁcation subsequently increased the portion of full- length templates in the assembly reaction to 34 and 29% for Taq and PfuTurbo Cx, respectively. This suggests that single base pair gaps generated after the excision of non- speciﬁc uracil residues and other lesions do occur, but ultimately do not impair the assembly process on a scale that would signiﬁcantly aﬀect the diversity of a library. The integrity of individual DNA templates was conﬁrmed by sequencing six clones of each purifying PCR (Figure 2, L4–6). Assembly substrates prepared with Taq DNA polymerase contained on an average one mutation in the protein-coding region comprising 624 base pairs. No mutations were found if assembly substrates were pre- pared with PfuTurbo Cx (Supplementary Figure S1, L4). In vitro TS–TL from assembled DNA templates To test whether the newly assembled DNA templates were functional for in vitro TS–TL, Avi-AGT was either expressed from DNA templates that have been ampliﬁed from assembly reactions or plasmid DNA (Figure 3). To detect functional Avi-AGT, the in vitro TS–TL reac- tion was supplemented with BG-biotin, a suicide inhibitor of AGT which irreversibly reacts with the active site cysteine of AGT and by that covalently labels the protein with biotin (27,30). No diﬀerence in the functional expres- sion of Avi-AGT could be detected between templates that have been ampliﬁed from assembly reactions or plasmid Figure 4. Assembly of an epPCR library. L1, hyperladder I in kilobase pairs;. L2, epPCR library prepared with the Genemorph II kit; L3, epPCR library of L2 re-ampliﬁed with Taq DNA polymerase and uracil-containing primers; L4, assembly of L3 with its UTRs+Avi- tag after it has been precipitated with PEG–MgCl2 and blunted with T4 DNA polymerase; L5, purifying PCR of L4 with 50 ng DNA per 100 ml PCR. Every sample lane contains 300 ng DNA. DNA (Figure 3). Similarly, the type of DNA polymerase made no diﬀerence in the functional expression levels either. Regarding the two bands that were observed in the functional expression test, we could exclude this to be a result of linker proteolysis since biotinylation of the Avi-tag with biotin ligase (as opposed to labelling of the active site with BG-biotin) similarly generated two distinct bands (Supplementary Data and Figure S2). Assembly of epPCR libraries To test the method under more demanding conditions, the procedure was applied to assemble epPCR libraries of AGT (Figure 4). epPCR libraries were initially prepared with the Genemorph II kit (Figure 4, L2). A portion of the library (50 ng equivalent to a diversity of 7 1010) was then re-ampliﬁed with Taq DNA polymerase to generate suitable assembly substrates with uracil residues near the 50-ends (Figure 4, L3). This additional step became neces- sary because ampliﬁcation with uracil-containing primers yielded no PCR product in the ﬁrst place (data not shown). Following precipitation with PEG–MgCl2 to remove residual primer dimers, the re-ampliﬁed library was assembled into the desired DNA template and puri- ﬁed over a short PCR run (Figure 4, L4-5). Sequencing of the library conﬁrmed the integrity of the library with an average of ﬁve mutations over the 574 bp that were subject to mutagenesis, and none of the clones containing fewer than three mutations. Assembly of DNA templates following model affinity selections We also demonstrate that the method can be applied to regenerate DNA templates following model aﬃnity