e122 Nucleic Acids Research, 2009, Vol. 37, No. 18 PAGE 4 OF 9 Figure 1. (A) Assembly Scheme. (i) GOI or a derivative library is ampliﬁed with primers that speciﬁcally incorporate uracil nucleotides close to both 50-ends. (ii) Assembly of the GOI with its 50- and 30-untranslated regions including any constant protein-coding regions based on a coupled uracil excision–ligation strategy. (iii) Pure templates are obtained following a short-purifying PCR, which eﬀectively ‘removes’ excess substrates and partially assembled intermediates. (B) Mechanism of coupled uracil excision–ligation: ﬁrst, USER enzyme catalyses the excision of uracil from DNA, thereby leaving a single base pair gap and a 30-extension provided the 50-portion can dissociate. Complementary overlapping 30-extensions then direct the assembly of DNA fragments which are covalently sealed by T4 DNA ligase. assembly substrates with T4 DNA polymerase (Supplementary Data and Figure S1). No further improvements could be achieved by lower- ing the temperature, higher quality oligonucleotides or increasing the reaction time and the amount of assembly substrates (data not shown). Similarly, it is unclear to what extent the assembled DNA templates are aﬀected by gaps caused by singly recessed 30-ends or non-speciﬁc uracil residues arising from oxidative damage such as heat induced conversion of cytidine to uridine (29). For instance, if templates are solely propagated by PCR in vitro, damaged DNA cannot be repaired by the bac- terial repair machinery following transformation in vivo (22–24). Gaps and other types of DNA damage could thus signiﬁcantly reduce the number of full-length DNA templates in vitro. This is not unlikely, especially if one considers that the assembly reactions performed here (Figure 2, L3 and Supplementary Figure S1) and else- where (22) were generally incomplete. The desired product template was, therefore, puriﬁed through a short PCR run over 10 ampliﬁcation cycles, which aﬀorded clean templates in 30 min (Figure 2, L4-6). A typical PCR contained a total of 50–500 ng DNA in a reaction volume of 100 ml. Given that the desired product contains 25–33% of all DNA (Figure 2, L3), this is equivalent to 1010–1011 template molecules and constitutes a suitable library size for the majority of in vitro screening and selection systems. Ampliﬁcation over more than 10 PCR cycles is not advisable as this may result in additional non-speciﬁc by-products, espe- cially primer dimers (data not shown). To exclude that Figure 2. Eﬃciency of template assembly by uracil excision–ligation and the purifying PCR for DNA templates prepared with Taq DNA polymerase. Precipitation with PEG–MgCl2 is necessary and suﬃcient for the eﬃcient assembly. L1, hyperladder I in kilobase pairs; L2, sub- strate AGT ampliﬁed with Taq, precipitated with PEG–MgCl2 and blunted with T4 DNA polymerase; L3, assembly of L2 with its UTRs + Avi-tag; L4, purifying PCR of L3 with 50 ng DNA per 100 ml PCR; L5, purifying PCR of L3 with 250 ng DNA per 100 ml PCR; L6, purifying PCR of L3 with 500 ng DNA per 100 ml PCR; L7, hyperladder I in kb; L8, substrate AGT ampliﬁed with Taq and blunted with T4 DNA polymerase; L9, assembly of L8 with its UTRs + Avi-tag; L10, purifying PCR of L9 with 50 ng DNA per 100 ml PCR. Every sample lane contains 300 ng DNA. the assembled DNA templates were signiﬁcantly aﬀected by single base pair gaps caused by non-speciﬁc uracil resi- dues, the assembly eﬃciency was additionally quantiﬁed by real-time PCR. For the purpose of quantiﬁcation, the