PAGE 7 OF 9 precipitation with PEG–MgCl2 is virtually unused in prac- tice. Our results emphasize that this step is critical and, if applied prior to the assembly step, overcomes the need to gel purify DNA templates after the assembly or purifying PCR steps: ﬁrst of all, it saves time and labour as precip- itation can be completed in 10–15 min as opposed to several hours needed to separate and extract DNA frag- ments from agarose gels. Furthermore, it prevents the GOI from becoming damaged following exposure to UV light during gel excision and pre-empts any inhibiting eﬀects that contaminants such as residual agarose and salts may have on downstream reactions (7,17,18). For instance, the majority of commercial in vitro TS–TL sys- tems explicitly advise not to purify DNA templates by agarose gel electrophoresis (17,18) Similarly, it has been reported in the literature that templates are not suﬃciently clean for in vitro TS–TL following excision from agarose gels using commercial puriﬁcation kits and generally need to be further puriﬁed by ethanol precipitation or include additional washing steps, e.g. with washing buﬀer during puriﬁcation with the QIAquick kit (7). Similarly, we found that the eﬃciency of PCR is markedly reduced if the DNA was gel puriﬁed prior to ampliﬁcation in the absence of additional puriﬁcation steps (data not shown). If a PCR is particularly prone to non-speciﬁc by- products that cannot easily be puriﬁed by precipitation with PEG–MgCl2, it may be necessary to perform two successive nested PCRs (12): a standard PCR to recover the desired DNA template followed by a short second run over 10 cycles to generate the assembly substrate with two uracil residues. This may particularly apply in real selec- tions where very few DNA fragments serve as templates and the ampliﬁcation eﬃciency of the target amplicon is compromised by greater competition from non-speciﬁc ampliﬁcation reactions. In terms of sequence requirements, template assembly through uracil excision–ligation only imposes minimal restrictions on the splice sites: only an adenine and a thy- midine spaced apart by several nucleotides in the 50!30 direction to ensure eﬃcient dissociation of the excised regions are required (22–24). In the context of directed evolution, the procedure allows rapid and ﬂexible varia- tion of the diversiﬁed regions saving laborious subcloning steps when diﬀerent regions of a gene are targeted and no additional restriction sites need to be introduced. In practice it should be considered that all splice sites need to diﬀer by at least two base pairs for a complementary overlap of 5–6 bp to prevent single-stranded extensions from cross-hybridizing (data not shown). Equally, it must be ensured that the single-stranded extensions cannot fold onto themselves to prevent the formation of covalently closed loops. Calculating the base pair prob- abilities of the single-stranded extensions with a suitable folding programme gives a good indication if secondary structures pose a problem, e.g. with the RNA-fold web server (http://rna.tbi.univie.ac.at/) which conveni- ently displays base pair probabilities as a dot plot (31,32). In comparison, template assembly procedures based on overlap extension PCR or restriction–digestion and liga- tion take signiﬁcantly longer—up to an entire day as opposed to 90 min. This can be attributed to sequential Nucleic Acids Research, 2009, Vol. 37, No. 18 e122 restriction–digestion and ligation reactions, and relatively long overlap extension and purifying PCRs featuring 25–30 cycles. Furthermore, the purifying PCR frequently generates non-speciﬁc fragments which require the desired DNA template to be puriﬁed by agarose gel electrophor- esis (7–10,12). The reason for the purifying PCR fre- quently being ineﬀective is presumably a consequence of the reduced eﬃciency of the overlap extension and ligation reactions. For instance in ligation reactions, template cir- cularization faces additional competition from template concatemerization. Furthermore, the palindromic nature of the single-stranded extensions generated by the major- ity of restriction enzymes enable DNA fragments to form homodimers which can be—just as the desired product— exponentially ampliﬁed by PCR when they are formed between the ﬂanking regions or the plasmid backbone. In addition, the eﬃciency of restriction–digestion and liga- tion strategies is often limited by the idiosyncrasies of indi- vidual restriction enzymes, which can cause unexpected problems such as ineﬃcient digestion at restriction sites located near the end of a linear DNA fragment (33). In overlap extension PCR, a qualitative analysis of DNA hybridization kinetics suggests that even under perfect conditions in the absence of any secondary structures only a fraction of templates recombines in a given thermal cycle. For instance, the hybridisation half lives for an overlap of 25 bp with both assembly substrates present at a concentration of 10 nM is on the order of 6–7 min (34,35). This compares to recombination times of 1 min that feature in most, if not all, overlap extension PCR protocols that have been published in textbooks (36,37), commercial product manuals (38) and scientiﬁc publica- tions that either speciﬁcally deal with the subject of over- lap extension PCR (39) or apply it in the context of directed evolution to assemble and regenerate libraries (9,10). In fact, it is unclear to what extent overlap exten- sion PCR is suitable to assemble gene libraries. For instance, templates that by chance recombine in an earlier thermal cycle will also enter exponential ampliﬁcation ear- lier, and thus increase in abundance so that templates are not uniformly ampliﬁed. If the size of a library then exceeds the recombination eﬃciency so that stochastic hybridization events cannot be averaged out for subpopu- lations of identical mutants, the library will become ran- domly biased. This makes the enrichment less dependent on a functional trait and may even reduce the diversity of a library as a portion of mutants can be eliminated during the assembly process. In some cases, the entire template has been ampliﬁed after selection so that template reassembly is unnecessary in the ﬁrst place (5,16). This strategy is, however, not broadly applicable; e.g. it has been reported that PCR ampliﬁcation is ineﬃcient if identical set of primers are used to prepare templates and recover genes after selection (7). This can be attributed to the partial, exonucleolytic degradation of the ends either by the 30!50 exonuclease activity of a proof-reading polymerase or due to any exo- nuclease activities in the cell extract (7,13). Successive nested PCRs of the whole template are equally impracti- cal; e.g. if synthetic modiﬁcations are introduced (5,9,27), each set of nested oligonucleotides needs to be modiﬁed