Biomedical Engineering Reference
In-Depth Information
restriction enzymes, one would be able to amplify the DNA fragment of an
expected size if the CpG dinucleotides within the recognition sequences are
methylated and, hence, resistant to enzyme digestion
(
12
)
. Using methylation-
insensitive restriction enzymes, no PCR products are obtained regardless of
the methylation status of CpG sites within the restriction sites. The major
limitation of this method is that the enzyme digestion must be complete
(
2
)
(
see
Note 6
). Another limitation is that if several CpG-rich restriction sites are
present in the target region (some CpG sites are methylated and some are
unmethylated), the results obtained can be inconclusive (
see
Note 7
).
3.4. Combined Bisulfite Restriction Analysis
After bisulfite modification and PCR, the conversion of unmethylated
cytosines to thymines and the retention of methylated cytosines can generate
new restriction sites or retain restriction sites such as “CGCG” for
Bst
UI
digestion
(
6
)
. The PCR products are thus sensitive to digestion by methylation-
sensitive restriction enzymes, which recognize and digest the methylated DNA
sequences. For COBRA, PCR primer sequences do not contain CpG dinucle-
otides, so PCR amplification does not discriminate between methylated and
unmethylated DNA templates. Therefore, the resulting PCR products may
consist of some DNA fragments with newly created or retained restriction sites
containing CpG dinucleotides, indicating the relative amounts of DNA
sequences with methylated CpG sites (digested PCR products) at particular
restriction sites in the DNA fragment
(
6
)
. COBRA is thus a quantitative method
for determining the methylation levels of particular CpG sites (
see
Note 8
).
3.5. Methylation-Sensitive Single Nucleotide Primer Extension
Quantitative assessment of DNA methylation changes at specific CpG sites
can be performed by Ms-SNuPE based on bisulfite modification of DNA and
single nucleotide primer extension
(
7
)
(
see
Note 8
). The single nucleotide
primer extension assay was first described by Kuppuswamy and others for the
detection of mutations in abnormal alleles
(
21
)
. Gonzalgo and Jones
(
7
)
modi-
fied this method for the quantification of DNA methylation differences at spe-
cific CpG sites. In contrast with COBRA, this method requires radioisotope
incorporation.
DNA is first treated with sodium bisulfite to convert unmethylated cytosines
to uracils, but methylcytosines remain as cytosines. After bisulfite treatment
and PCR amplification of the target sequence with gene-specific primers, PCR
products serve as templates for MS-SNuPE. The primers used for the single
nucleotide extension are designed so that the primer ends immediately 5' of the
single nucleotide (just before the incorporation site) designated for the methy-
lation analysis.
