Biomedical Engineering Reference
In-Depth Information
different, ranging from 10
-5
to
10
-2
(
9
,
10
,
17
,
18
)
. The lower detectability of me-
thylation changes may possibly be related to the lower sensitivity of the MSP
assay. The detectability of methylation changes can possibly be further
enhanced if a larger amount of DNA is added for PCR. Highly specific and
sensitive MSP should be applicable for the methylation analysis among the
low percentage of target cells and particularly useful for detecting small
amounts of methylated target alleles.
3.1.1. DNA Extraction
The quality of DNA extracted from different sample sources is the deter-
mining factor affecting the accuracy of DNA methylation analysis. Also, the
DNA quantity should be considered when determining which PCR-based
method should be used for the methylation analysis. In particular, when the
DNA amount is less than 1
g, carrier DNA should be added to the DNA solu-
tion before the bisulfite modification steps.
µ
3.1.2. Bisulfite Modification of DNA (see
Note 5
)
1.
Add 7.0
µ
L of 3
M
sodium hydroxide to 1
µ
g DNA in 100
µ
L of autoclaved
distilled water and incubate the mixture at 37°C for 10 min.
2.
Add 550
L of freshly prepared DNA Modification Reagent I (CpGenome DNA
modification kit; Chemicon International Inc.) and vortex.
µ
3.
Incubate at 50°C for 16-20 h in a heat block or water bath.
4.
Add 5
L of well suspended DNA Modification Reagent III from the kit to the
incubated reaction mix.
µ
5.
Add 750
µ
L of DNA Modification Reagent II and mix briefly.
6.
Centrifuge at 5000
g
for 10 s.
7.
Add 0.5 mL of 70% ethanol and vortex.
8.
Centrifuge at 5000
g
for 10 s and discard the supernatant.
9.
Repeat the wash with 70% ethanol for two more times.
10.
Remove the supernatant and dry the sample for 5 min.
11.
Resuspend the pellet with 50
L of 20 m
M
sodium hydroxide/90% ethanol solu-
tion and incubate at room temperature for 5 min.
µ
12.
Centrifuge at 5000
g
for 10 s and remove the supernatant.
13.
Add 1 mL of 90% ethanol and vortex.
14.
Centrifuge at 5000
g
for 10 s and remove the supernatant.
15.
Repeat
steps 10
and
11
.
16.
Dry the DNA pellet and dissolve in 25
µ
L of TE buffer.
17.
Incubate the sample at 50-60°C for 5 min to elute the DNA.
18.
Centrifuge at 5000
g
for 10 s and collect the supernatant for storage at -20°C.
3.1.3. PCR Primers
As an example, the methylated and unmethylated primer sequences for ana-
lyzing
p15
methylation by MSP are listed in
Table 2
(
10
)
.
