20 nm peak-to-peak, resulting in a low stored energy that increased the measurement sensitivity. Gel electrophoresis The products of the polymerase synthesis were analyzed by 0.7% agarose gel electrophoresis at room temperature at 130 V for 1 h. TAE buﬀer was used for the agarose preparation and as a running buﬀer. The dimensions of the agarose gel were 10 10 cm with 2 4 mm 14-wells. The gel was stained with ethidium bromide (0.5 mg/ml) and visualized with a Bio Imaging System (302 nm). RESULTS Preparation and characterization of the avidin–Poly(dG)-Poly(dC) complexes We have synthesized double-stranded poly(dG)-poly(dC) molecules composed of C-homopolymers and 50-end biotin-labeled G-homopolymers, as described in ‘Materials and methods’ section. The polymerase synthe- sis, conducted essentially as described in (39) yielded 1.4 kb 50biotin–poly(dG)-poly(dC) molecules character- ized by a narrow length distribution (Figure 1, lane 2). Incubation of the synthesized 50biotin–poly(dG)- poly(dC) molecules with the avidin-tetramer as described Nucleic Acids Research, 2008, Vol. 36, No. 15 5053 in ‘Materials and methods’ section, results in the forma- tion of the complex between the DNA and the protein. Figure 1 shows gel electrophoretic patterns of complexes obtained during incubation of 50biotin–poly(dG)-poly(dC) with avidin at diﬀerent DNA to avidin ratios. As can be seen in the Figure 1, incubation of the avidin-tetramer with twice the amount of 50biotin–poly(dG)-poly(dC) yields a complex with a mass of 3 kb (Figure 1, lane 3) and thus corresponds to two 50biotin–poly(dG)-poly(dC) molecules joined together by avidin. The increase of the ratio between the DNA to the protein leads to the forma- tion of higher molecular mass ( 5 and 6 kb) complexes corresponding to three and four 50biotin–poly(dG)- poly(dC) molecules linked together (Figure 1, lanes 4–6), respectively. Increasing the ratio above four results in a mixture of the saturated avidin–DNA complex, com- prising of four 50biotin–poly(dG)-poly(dC) molecules (tetra-molecular complex), and single 50biotin–poly(dG)- poly(dC) molecules not grouped by avidin (Figure 1, lane 6). A minor quantity of the tri-molecular complex is pre- sent together with the tetra-molecular one even in the mixture containing a large (5 and more) excess of DNA over the protein (Figure 1, lane 6). The tetra-molecular complex prepared at a 5 : 1 ratio between 50biotin– poly(dG)-poly(dC) and avidin (Figure 1) was puriﬁed from the excess of unbound DNA using size-exclusion HPLC. The elution proﬁle (Figure 2A) shows two major peaks. The ﬁrst one, eluted between 12 and 14 min, corre- sponds to the avidin–4[biotin–poly(dG)-poly(dC)] com- plex; and the second peak, eluted between 14 and 16 min, to 50biotin–poly(dG)-poly(dC) not linked to avidin. The molecular composition and the purity of molecules eluted in the ﬁrst peak were analyzed by agarose gel-electrophoresis (Figure 2B). The electrophoretic analy- sis clearly shows that the complex is completely separated Figure 1. Mobility of poly(dG)-poly(dC)–avidin complexes in 0.7% Agarose gel. A 1.4 kb 50biotin–poly(dG)-poly(dC) (lane 2) was incu- bated with avidin at molar ratios of 2, 3, 4 and 5, as described in ‘Materials and methods’ section—lanes 3, 4, 5 and 6, correspondingly. Lane 1 is 1 kb DNA Ladder. Figure 2. HPLC puriﬁcation of avidin–50biotin–poly(dG)-poly(dC) complex. (A) Size-exclusion HPLC separation of avidin–50biotin– poly(dG)-poly(dC) complex from unbound 50biotin–poly(dG)- poly(dC). The separation was carried out on the G-DNA PW column in 20 mM Tris–Acetate buﬀer, pH 7.4 and 0.5 M NaCl, as described in ‘Materials and methods’ section. (B) 0.7% agarose gel electrophoresis of the puriﬁed avidin–50biotin–poly(dG)-poly(dC). The complex was eluted from the column between 12 and 14 min.