Biomedical Engineering Reference
In-Depth Information
10.4
Direct Detection of DNA Methylation
by “Southern- Western” Blotting
We estimated and compared the methylation levels in genomic DNA from mouse,
Drosophila
, and honeybee brains by “Southern-Western” blotting. Using anti-5-
methylcytosine mouse antibody easily enabled to combine Southern blotting and
Western blotting.
10.4.1
Procedure for Southern-Western Blotting
To compare the relative amount of whole-genome methylation, 5
g of honeybee,
mouse, and
Drosophila
genomic DNA were run on a 0.8 % agarose gel and trans-
ferred onto a nitrocellulose membrane following a standard method for Southern
blotting. After treatment with a blocking buffer containing 1 % BSA and 1 %
Tween-20 in phosphate-buffered saline (PBS) for 1 h at room temperature, the
membrane was incubated with anti-5-methylcytosine mouse antibody (Calbiochem)
diluted to 1:1,000 with blocking buffer and incubated overnight at 4 °C. Other
blocking buffers, such as 5 % skim milk and 1 % Tween-20 in PBS, could have also
been used for this experiment. The membrane was rinsed with washing buffer
(0.1 % Tween-20 in PBS; 3 × 15 min) and incubated with horseradish peroxidase
(HRP)-labeled anti-mouse IgG diluted to 1:10,000 with blocking buffer at room
temperature for 1 h. The membrane was then rinsed with washing buffer (3 × 15 min)
and treated with chemiluminescent HRP substrate for 5 min at room temperature.
μ
10.4.2
Results of Southern-Western Blotting
Although equal amounts of genomic DNA (5
g) purifi ed from honeybee,
Drosophila,
and mouse brains were subjected to agarose gel electrophoresis
(Fig.
10.2a
), the intensity of the band from the honeybee genomic DNA was lower
than that from mouse and
Drosophila
genomic DNA (Fig.
10.2b
). This data sup-
ports a previous report that found a smaller amount of methylcytosine in honeybee
and
Drosophila
genomic DNA than mammalian DNA (Wang et al.
2006
).
μ
10.5
Methylation-Sensitive Restriction
Besides the enrichment of methylated DNA by immunoprecipitation (which is
pretty much dependent of local CpG content and DNA accessibility), methylation-
specifi cally cutting endonucleases can be used for investigating methylation
