Supercoiling Assay

The disruption of chromatin structure can be also detected by supercoiling assay. This assay is based on the observation that in vitro the assembly of each nucleosome into a circular plasmid DNA will introduce one negatively super-helical turn (21). If a transcription factor has the capacity to exert disruption of chromatin, it will result in the change of the number of superhelical turns. The plasmid molecules with different superhelical turns can be resolved in agarose gel containing chloroquine.

1. Make a 1-1.2% agarose gel in 1X TPE buffer containing 90 pg/mL of chloroquine diphosphate (Sigma; cat no. C-6628). Add chloroquine when agarose solution is not very hot (approx 70°C).

2. Prepare 1 L of 1X TPE buffer with 90 pg/mL of chloroquine.

3. Add agarose gel loading buffer without bromophenol blue to DNA samples recovered from injected oocytes (see Subheading 3.3.3. DNA Preparation) (bro-mophenol blue forms precipitates with chloroquine). As a marker, xylene cyanole FF (0.01%) can be added to the gel loading buffer. As experienced, DNA recovered from 2 to 3 injected oocytes is sufficient for supercoiling assay detected by Southern hybridization.

4. Load DNA samples to gel. Circulate TPE electrophoresis buffer using a Pharmacia P-1 pump with a high flow rate. It is necessary to circulate the buffer because the TPE buffer has a low capacity. Cover the gel apparatus with aluminum foil or run the gel in the dark (chloroquine is light-sensitive). Run the gel at 35 V overnight (it depends on the size of the plasmid analyzed. For a 4.5-kb TR0A construct, it take about 12-14 h).

5. After electrophoresis, wash the gel twice in 500 mL water for 1 to 2 h to remove chloroquine.

6. Treat the gel with 500 mL of 0.25 M HCl for 30 min.

7. Rinse the gel with water and then treat with 0.5 M NaOH, 1.5 M NaCl for 30 min, followed by treatment with 0.5 M Tris-HCl, pH7.5, 1.5 M NaCl for 30 min.

8. Transfer DNA to a piece of nylan membrane with 20X standard saline citrate (SSC) according to "Current Protocol in Molecular Biology" (20).

9. Supercoiling pattern can be detected with any probes derived from the constructs to be analyzed (Note 12).

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