Single molecule techniques to measure biological molecules and reactions have provided an alternative way to probe and visualize bond characteristics and reaction dynamics. However, these techniques, such as atomic force microscopy, optical tweezers, and micropipettes often require expensive and complicated equipment and are very time intensive, because each measurement gives the results of one-single reaction or a property of one-single molecule. Here, we report on a technique that allows for massively parallel measurements on many individual single molecules in microfluidic systems. We demonstrate the effectiveness of a simple, robust, inexpensive apparatus, by using it to differentiate between deoxyribonucleic acid (DNA) assemblies that are merely annealed from others that are ligated, and by measuring the rate at which annealed DNA denatures as function of temperature.

Topics
Optical tweezers, Microfluidic devices, Atomic force microscopy, Deoxyribonucleic acid, Single molecule techniques, Biomolecules
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The biotinylated oligonucleotides (“left-hand side oligo:” 5′-Agg TCg CCg CCC TTT Biotin-3′, “right-hand side oligo:” 5′-ggg Cgg CgA CCT TTT Biotin-3′, “poly T:” 5′-TTT TTT TTT TTT TTT Biotin-3′, Oligos Etc. Inc.) were attached to the streptavidin-coated beads by mixing in 5 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 1.0 M NaCl buffer solution during 15 min at room temperature with five-fold excess. After washing and transferring the beads to the working buffer (1×E.coli Ligase Buffer, GIBCO BRL), the oligonucleotides (designed with the correspondent 12-base pair of each single-stranded λ-DNA end) are ready to bind the DNA by hybridization.
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The preparation is done sequentially, using the same oligos as for the tethered case. DNA and left-hand side oligo (1:30 molar ratio) were hybridized (55 °C, 1 h) and then ligated (T4 ligase, 16 °C, 1 h). Toward this preparation, the right-hand side oligo was hybridized (1:100 molar ratio, 55 °C, 1 h) and then ligated (T4 ligase, 16 °C, 1 h) and finally purified (Amicon 100 centrifugal filter, Millipore).
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A settling time of 5 min was choosen for the 200 μm thick microchannels in order to allow all beads to reach the glass surface. The DNA and the bead concentration was 4×10−10 M and 2.41×104/μl, respectively.
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After the streptavidin-coating procedure the surface was incubated for 30 min with a 0.1 mM biotin solution in 1×E.coli Ligase Buffer.
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2002
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