Biomedical Engineering Reference
In-Depth Information
Quantification of the ratio of methylated cytosines vs thymines (unmethy-
lated cytosines) at the original CpG sites can be determined by incubating the
gel-purified PCR products, primers, and
Taq
polymerase with either [
32
P] dCTP
or [
32
P] dTTP followed by denaturing polyacrylamide gel electrophoresis and
phosphorimage analysis. Opposite-strand Ms-SNuPE primers that would in-
corporate either [
32
P] dATP or [
32
P] dGTP to assess the methylation status of
individual CpG sites can also be designed. If the target CpG site is methylated,
a C is incorporated during nucleotide extension. If the CpG site is
unmethylated, a T is incorporated instead. Quantification of the incorporated C
or T determines the methylation status of the particular CpG site. As discussed
previously, primer design and complete modification of DNA are important in
order to obtain consistent results.
3.6. Quantitative Real-Time MSP
Using a quantitative approach (
see
Notes 3
and
8
), the biological implica-
tions of DNA methylation changes can be clarified. Previously, real-time quan-
titative MSP has been developed to demonstrate the biological significance of
p16
methylation index
(
8
,
22
)
.
In this system, two amplification primers and a
dual-labeled fluorogenic hybridization
probe are included. One fluorescent dye
serves as
a reporter (fluorescein [FAM]), and its emission spectra are quenched
by a
second fluorescent dye (tetramethylrhodamine [TAMRA]). During the
extension phase of
PCR, the 5' to 3' exonuclease activity of the
Taq
DNA poly-
merase
cleaves the reporter from the probe, thus releasing it
from the quencher,
resulting in an increase in fluorescent emission
at 518 nm.
Three real-time MSP
systems are required for the quantification of the bisulfite-converted methy-
lated sequence, the bisulfite-converted
unmethylated sequence, and the uncon-
verted wild-type sequence of the target gene. DNA amplification can be carried
out
in a 96-well reaction plate format in an Applied Biosystems
7700 Sequence
Detector.
Calibration curves are set up in parallel with each analysis using ref-
erence DNA samples with known methylation status.
The methylation index (%) in a sample is calculated using the
following
equation:
M
M+U
×
where
M
is the quantity
of methylated sequences measured by real-time MSP
after bisulfite conversion and
U
is the quantity of unmethylated
sequences.
The
usefulness of this fluorescence-based real-time MSP has also been validated
by two other groups who aimed at the quantification of
p16
and
hMLH1
methylation changes
(
23
,
24
)
.
Methylation index =
100%
