PAGE 3 OF 8 for 1–3 min at 10 000 rpm. The sample was then eluted in 20 ml ddH2O and used for further experiments. Removal of the free STV by gel puriﬁcation was done by loading the sample onto an agarose gel, excising the A+STV frag- ment and cutting the gel slice into small pieces, which were then centrifuged for 15 min at 14 000 rpm (38). The supernatant containing the A+STV fragments was after- wards collected and used in the second incubation step. Although this technique for gel extraction has a relatively low recovery for long molecules, it was chosen because the nucleic acid–STV complex does not survive the buﬀers used in most commercial gel extraction kits. Other techni- ques either require high temperatures and/or ethanol pre- cipitation, which destabilize the nucleic acid–STV complex. We note that extraction of the nucleic acid– STV complex using electro-elution might also be a viable alternative, with possibly higher recovery. Gel analysis Samples containing molecules up to 10 kb in length were all analyzed by gel electrophoresis in nondenaturing 0.8% agarose gels in TAE buﬀer at 5 V/cm. DNA samples of longer molecules were analyzed by pulsed ﬁeld electrophor- esis (CHEF Mapper, Bio-RAD technologies, Hercules, CA, USA) on 1.0% agarose gels in 0.5 TBE buﬀer using a program optimized for separating DNA molecules between 10 and 80 kb. All agarose gels were stained with ethidium bromide (EtBr). Atomic Force Microscope (AFM) images The gel-extracted heterodimers of 2.2 kb+3.5 kb DNA were imaged with tapping-mode AFM (National Instru- ments, Austin, TX, USA, Nanoscope IV) in air by deposit- ing 5 ml of 1 ng/ml of DNA solution on freshly cleaved mica. After 2 min, the sample was rinsed for 10 s with ddH2O water, blown dry with N2, and directly imaged in the AFM. The AFM image in Figure 1c was analyzed using the freeware WsXM (39). Determination of binding efficiency The eﬃciency of the joining reactions is determined as follows: the intensities of the bands corresponding to the biotinylated molecules A+STV, B+STV, A+B, A+A and B+B were determined using the program LabImage 1D (Kapelan GmbH, Germany) and each was divided by the respective molecular weight to obtain a relative molar amount NA+STV, NB+STV, NA+B, NA+A and NB+B. From this calculation, we determined which of the reactants A or B was limiting (i.e. which reactant was present in the lowest amount before the reaction). The eﬃciency of the reaction was then calculated as follows: eﬃciency (A+B) = NA+B/(NA+STV+NA+A+NA+B) in the case of limiting reactant A, and eﬃciency (A+B) = NA+B/(NB+STV+NB+B+NA+B) in the case of limiting reactant B. We do not take into account the A and B molecules that did not bind STV, because these represent molecules that were not biotinylated and there- fore do not participate in the reaction. Depending on the application, the ﬁnal product itself may require further puriﬁcation, which, depending on the protocol employed, Nucleic Acids Research, 2008, Vol. 36, No. 16 e104 could additionally aﬀect the overall eﬃciency [e.g. the gel puriﬁcation method described above has a typical recov- ery of 50–80% (38)]. RESULTS Formation of DNA–STV–DNA heterodimers In our experiments, we aim to optimize the speciﬁc for- mation of the dimer A+B starting from unlinked mole- cules A and B. To achieve this, we perform a two-step reaction, shown schematically in Figure 1a. In the ﬁrst step, molecule A is incubated in buﬀer 1 for 30 min together with a large excess of STV (>30 molar excess). Due to the excess STV, all biotinylated molecules will rapidly be bound to single STV molecules. Because these A+STV complexes cannot bind to each other (they can only bind to those A molecules in which the biotin remains unbound), this has an additional advantage that it almost completely suppresses the formation of homodi- mers A+A. After this ﬁrst step, the free STV is removed by gel puriﬁcation and the second nucleic acid molecule B is added in approximately a 1 : 1 molar ratio to molecule A. This is incubated at 48C for a duration of 30 min up to several hours. Provided that all free STV has been success- fully removed after the ﬁrst incubation, the B molecules exclusively bind to the STV-tagged A molecules, resulting in the formation of heterodimers A+B. The result of this protocol with two monobiotinylated DNA molecules of lengths 3.5 kb (molecule A) and 2.2 kb (molecule B) is shown in Figure 1b. For reference, Lane M contains a DNA marker. In lane 2 we show the result after following the protocol without STV, which as expected shows clear bands corresponding to 2.2 kb DNA and 3.5 kb DNA. In lane 1, molecules A and B were incubated according to the protocol described above. The clear band at 5.7 kb (circled) corresponds to the dimer A+B. An AFM image of the resulting A+B dimer deposited on mica is shown in Figure 1c: here, we can clearly distin- guish two DNA molecules joined at the ends via a slightly higher intermediary. The intermediary is likely the STV, as STV is signiﬁcantly larger (4–6 nm diameter) than the DNA (2.2 nm diameter) and should therefore appear higher in the AFM (40) (note that the apparent height of both the DNA and the STV in the AFM is lower, because the AFM tip deforms the DNA and STV as is commonly observed in AFM imaging). The height proﬁle of a trace along the contour of the construct is taken to conﬁrm the lengths: we ﬁnd the distances along the DNA to the STV are about 1200 50 nm and 750 50 nm. This matches well with the expected length values for molecule A (3524 bp 0.34 nm/bp = 1192 nm) and molecule B (2250 bp 0.34 nm/bp = 765 nm). We now investigate the bands in Figure 1b, lane 1, that do not correspond to the desired end-product dimer A+B. These bands migrate at approximately the same speed as the unbound molecules A and B in lane 2. However, closer inspection reveals a slight shift in the band corresponding to molecule A. Because this shift only appears in the pre- sence of STV, it can be attributed to molecules A that are bound to STV, and is observable due to the fact that the