Methylation specific PCR

Control DNA

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

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