Purification and Hybridization of Labeled Oligonucleotides

3.2.1. Polyacrylamide Gel Electrophoresis

Quantitative FRET measurements to determine dye-to-dye distances and to do structural prediction of nucleoprotein complexes require a high quality of the oligonucleotides, dsDNA, and a high labeling efficiency. Thus, all partly present shorter sequences and unlabeled oligonucleotides must be removed. To achieve this purify oligonucleotides and dsDNA by PAGE. Even when the oligonucleotides were purified by reverse-phase high-performance liquid chromatography this additional purification step is recommended.

1. Clean glass plates (40 x 20 cm) with 70% ethanol and let air-dry. Assemble the sandwich using a 1.5-mm spacer between the glass plates and seal the bottom with tape.

2. Prepare 150 mL of polyacrylamide solution from a 30% PAGE stock solution of the following concentration depending on the length of the oligonucleotides:

up to 20 nucleotides18% 20-30 nucleotides 16% 30-40 nucleotides 14% 40-50 nucleotides 12% 50-60 nucleotides 10%

3. Add 63 g urea (7 M) for denaturing conditions and finally add 1.4 mL of 10% ammonium persulfate and 70 ||L TEMED while stirring on ice. Generate the polyacrylamide gel by avoiding air bubbles and let the acrylamide solution polymerize for 30 min up to 1 h. Use a 1.5-mm thick sample comb with approx 3-cm long sample chambers.

4. Use 1X TBE as electrophoresis buffer and pre-run electrophoresis for 1 h at 30 W (max 600 V). Use a 3-mm thick aluminum plate in front of the top glass plate for a homogenous heat transport. Performing electrophoresis under these conditions produces a lot of heat and would otherwise result in broken glass plates.

5. Denature approx 50 ||L of oligonucleotide solution (0.1 nmol/|L) for 5 min at 90°C in a heating block and add 10 |L of 6X DNA loading buffer.

6. After equilibration of the acrylamide gel, remove the excess of urea from the sample chambers and load the preheated oligonucleotides.

7. Run electrophoresis for approx 5-8 h at 30 W (max 600 V) and protect the samples from light by placing a box above the apparatus.

8. Isolate the oligonucleotides by electroelution as described in Subheading 3.2.2.

9. Transfer the sample from the high-salt elution buffer into H2O using size-exclusion chromatography (see Subheading 3.2.3.).

10. Determine oligonucleotide concentration and label efficiency by UV/Vis spectroscopy (see Subheading 3.3.1.).

11. Lyophilize the samples using a SpeedVac and store the samples at -20°C.

3.2.2. Isolating Oligonucleotides by Electroelution

1. Fluorescently labeled oligonucleotides can be visualized by UV-illumination in the dark room and cut out of the gel with a clean razor blade; nearly all fluorescent dyes absorb UV light.

2. Nonfluorescent samples can be visualized by DNA-shadowing. A thin-layer chro-matography plate with fluorescent indicator (254 nm) is laid under the gel and illuminated with UV light from a hand-held lamp. At spots where DNA is present a dark band can be seen; DNA absorbs at 260 nm resulting in a nonfluorescent band.

Fig. 1. Scheme of the electroelution apparatus. DNA moves along the electrical field from the gel into the V-channel where it gets trapped in the high salt buffer (3 M NH4Ac).

3. Fill the electroelution apparatus with 0.5X TBE buffer and place the gel slices in position (see Fig. 1).

4. Use a long-tip pipet to fill carefully 100 |L of 3 M ammonium acetate solution into the V-slit avoiding air bubbles (see Note 2).

5. Run the elution with a constant voltage of 100 V for approx 30 min in the dark (cover the apparatus with aluminum foil).

6. Collect the solution of the V-channel containing the oligonucleotides or dsDNA by using a 200-|L pipet. Repeat this step to collect as much as possible of the extracted DNA.

3.2.3. Buffer Exchange by Size-Exclusion Chromatography Using Spin-Columns

1. Centrifugate a Sephadex G-25 spin column (see also Subheading 2.1.) at 1600g for 4 min until it is dry (see Note 1).

2. For a buffer exchange equilibrate the spin column with the final buffer by adding 500 pL of the buffer to the dry column and spin again at 1600g for 4 min. Repeat this step three to four times.

3. Add oligonucleotide containing free dye or dsDNA solution (approx 400 | L) to the dry column and place a new 1.5-mL tube below the outlet of the column (see Note 1).

4. Centrifugate at 1600g for 4 min.

5. The final volume is approx 400 |L and contains 80-90% of the initial oligonucleotide or dsDNA.

6. Used columns can be restored by washing with an excess of H2O (approx 30 mL) and used several times.

3.2.4. Hybridization

1. Dissolve lyophilized oligonucleotides in hybridization buffer with a concentration of 40 |M.

2. Prepare three dsDNA samples: (1) donor only sample (D+/A-), (2) acceptor only sample (D-/A+), and (3) FRET sample containing both dyes (D+/A+). Mix 25 |L of each of the corresponding labeled and nonlabeled oligonucleotides.

3. Heat samples above melting temperature (80-90°C) in a heating block. Turn off the block and let the samples slowly cool down to room temperature overnight remaining in the block.

4. Separate the dsDNA from single-stranded samples by native polyacrylamide gel electrophoresis with 100 V for 1-2 h. A 10- to 20-cm long electrophoresis apparatus is sufficient. Prepare the acrylamide solution as described in Subheading 3.2.1. without the addition of urea.

5. Isolate the dsDNA samples from the gel by electroelution (see Subheading 3.2.2.) and change the buffer, e.g., protein binding buffer, by size-exclusion chromatog-raphy as described in Subheading 3.2.3.

6. Determine DNA concentration and label efficiency by UV/Vis spectroscopy (see Subheading 3.3.2.).

7. Store DNA in solution at 4°C until used.

0 0

Post a comment