Biomedical Engineering Reference
In-Depth Information
dinucleotides 0.65. The CpG islands of an increasing number of human genes
are known to be differentially methylated in human tissues.
DNA methylation can be easily analyzed by qualitative or quantitative poly-
merase chain reaction (PCR)-based methods
(
2
)
, including methylation-
specific PCR (MSP)
(
3
)
, bisulfite sequencing
(
4
,
5
)
, methylation-sensitive
restriction enzyme PCR, combined bisulfite restriction analysis (COBRA)
(
6
)
,
methylation-sensitive single nucleotide primer extension (Ms-SNuPE)
(
7
)
, and
quantitative real-time MSP
(
8
)
.
Table 1
enumerates the advantages and disad-
vantages of these various techniques for DNA methylation analyses.
MSP, which couples the bisulfite modification of DNA and PCR, is fast,
highly sensitive, relatively simple, inexpensive, and widely applied for DNA
methylation analyses. Bisulfite modification converts unmethylated cytosine
to uracil, whereas methylcytosine remains unmodified
(
4
,
5
)
. MSP requires spe-
cific primer sets, which are designed to distinguish between methylated and
unmethylated DNA sequences. MSP offers high sensitivity for detecting small
amounts of methylated alleles in clinical samples, such as plasma, serum, blood
cells, lymph nodes, biopsies, and paraffin-embedded tissues
(
9-11
)
.
Bisulfite
sequencing is comparatively time-consuming. Large-scale
sequencing of multiple plasmid clones is required in order to obtain the overall
methylation pattern
(
4
,
5
)
. Methylation-sensitive restriction enzyme PCR
combines methylation-sensitive restriction enzyme digestion and PCR
(
12
)
.
After enzyme digestion, PCR products are obtained if the enzyme does not
digest at the methylated CpG sites within the specified DNA region. COBRA,
Ms-SNuPE, and quantitative real-time MSP allow the quantitative analyses of
DNA methylation. However, these methods are rather costly or are not easily
established.
2. Materials
2.1. MSP, Methylation-Sensitive Restriction Enzyme PCR, COBRA,
MsSNuPE, and Quantitative Real-Time MSP
1.
Qiagen DNA extraction kit (Qiagen, Hilden, Germany).
2.
CpGenome DNA modification kit (Chemicon International Inc., Temecula, CA)
including sodium bisulfite reagents and carrier DNA.
3.
Sodium hydroxide.
4.
β
-mercaptoethanol.
5.
TE (Tris + ethylenediamine tetraacetic acid [EDTA]) buffer.
6.
Ethanol.
7.
Reaction buffers for PCR, restriction digestion and primer extension.
8.
Primers (
see
Note 1
).
