Qualitative and Quantitative Polymerase Chain Reaction-Based Methods for DNA Methylation Analyses - Clinical Applications of PCR - page 38

Table 2
Methylated and Unmethylated Primer Sequences for Analyzing p15
Methylation by Methylation-Specific Polymerase Chain Reaction

Methylated p15MF 5' GCG TTC GTA TTT TGC GGT T 3'

Methylated p15MR 5' CGT ACA ATA ACC GAA CGA CCG A 3'

Unmethylated p15UF 5' TGT GAT GTG TTT GTA TTT TGT GGT T 3'

Unmethylated p15UR 5' CCA TAC AAT AAC CAA ACA ACC AA 3'

3.1.4. PCR Conditions and Thermocycling Profiles
PCR amplification is carried out using reagents supplied in the GeneAmp
DNA Amplification Kit and Ampli Taq Gold polymerase. The thermal profile
consists of an initial denaturation step of 95°C for 12 min followed by repeti-
tions of 95°C for 45 s, 60°C for 45 s, and 72°C for 1 min, with a final extension
step of 72°C for 10 min ( 9 ) . A total of 35-45 cycles are generally used.

3.1.5. Molecular Analyses of PCR Products
PCR products are analyzed by gel electrophoresis using 6% polyacrylamide
gel or 2% agarose gel stained with ethidium bromide. Each DNA sample should
be analyzed at least in duplicate.

3.2. Bisulfite Sequencing
Bisulfite sequencing was first described by Frommer and others in 1992 ( 5 ) .
Bisulfite-modified DNA is PCR-amplified, cloned, and then sequenced.
Bisulfite sequencing of DNA containing CpG sites is an excellent method for
analyzing the methylation status of each individual CpG site within the target
sequence ( 4 ) . The method is based on the chemical modification of
unmethylated cytosine to uracil by sodium bisulfite; methylated cytosine
remains unmodified ( 19 ) . The modified DNA is then PCR-amplified using
gene-specific primers that anneal to the DNA sequences without CpG sites. In
other words, the primers anneal to the DNA sequences containing uracils in
positions of cytosines after bisulfite conversion. The amplified DNA fragments
can be sequenced after cloning of individual molecules ( 20 ) . It is very impor-
tant to verify whether the bisulfite conversion is complete.

3.3. Methylation-Sensitive Restriction Enzyme PCR
Digestion of DNA with methylation-sensitive restriction enzymes is
followed by PCR amplification using primers flanking the target region where
the restriction sites are located. DNA is cleaved with methylation-sensitive or
methylation-insensitive restriction enzymes. Using methylation-sensitive