Materials

1. Oligonucleotides: all oligonucleotides are purchased from a commercial source (e.g., Integrated DNA Technologies, Coralville, IA). The oligonucleotides are purified by high-performance liquid chromatography or polyacrylamide gel elec-trophoresis to assure high DNA purity.

2. Buffers: to prepare 500 mM of Tris-acetate buffer stock solution, add acetic acid (glacial) to 500 mM of Tris solution until pH drops to the desired value (pH 7.2 is used in the current system). Incubate the buffer stock solution with metal chelat-ing resin (iminodiacetic acid, sodium form) (Aldrich, St. Louis, MO) overnight to eliminate trace divalent metal ions. Finally, filter the buffer stock solution with a 0.2 |im syringe filters (Nalgene, Rochester, NY) and store it in a -20°C freezer.

3. Polyacrylamide gel electrophoresis reagents: boric acid, EDTA, ammonium persulfate (APS), TEMED, acrylamide, and bisacrylamide are purchased from

AT c

G C-TMR

G Enzyme strand

Fisher (Fair Lawn, NJ). Tris and urea are purchased from USB Corporation (Cleveland, OH). Prepare a 25% APS solution and store both the APS and TEMED in a refrigerator at 4°C.

4. Fluorometer: fluorescence emission spectra are recorded on an SLM 8000S fluo-rometer (ISS, Champaign, IL) operating in photon counting mode and corrected for lamp fluctuation and instrumental variations. The monochromator in fluo-rometers can distort fluorescence spectra because of polarization-dependent transmission. To avoid this artifact, set emission polarizers in the "magic angle" conditions, i.e., at an angle of 54.7° with respect to the direction of the excitation polarizer (15). Use a water bath to control temperature at 4°C and pump N2(g) into the cuvet holder area to prevent water condensation.

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