Methylation specific PCR
Control reactions should be performed when undertaking methylation analysis, for example, when performing methylation-specific PCR (MSP), to ensure that the PCR primers are specific for the detection of methylated or unmethylated bisulfite converted DNA.
To perform control reactions, methylated bisulfite converted DNA, unmethylated bisulfite converted DNA, and genomic DNA are required. Each type of DNA should be used to show the specificity of the PCR. For example, the primer specific for methylated DNA will only show a signal for the methylated control DNA (see table Expected PCR results with controls).
In addition, genomic DNA can be used to determine the bisulfite conversion efficiency of bisulfite reactions.
Expected PCR results with controls
|Type of DNA|| Primer for unmethylated
target gene (PCR 1)
| Primer for unmethylated
target gene (bisulfite converted) (PCR 2)
| Primer for methylated target
gene (bisulfite converted) (PCR 3)
|Unmethylated control DNA||PCR product||No PCR product|| No PCR product
|Unmethylated control DNA (bisulfite converted)||No PCR product||PCR product|| No PCR product
|Methylated control DNA (bisulfite converted)||No PCR product||No PCR product|| PCR product
|No-template control||No PCR product||No PCR product|| No PCR product