Specificity of Methylation Assays in Cancer Research: A Guideline for Designing Primers and Probes

Figure 1

Primer design for DNA methylation profiling techniques based on bisulfite conversion. (a) First DNA is treated with sodium bisulfite to convert all unmethylated cytosines to uracil. To analyze DNA methylation status of the interest genes, converted DNA is amplified based on two different primer designing strategies: methylation-independent specific PCR (MIP) and methylation-specific PCR (MSP). (b) In MIP, DNA molecules are amplified using primer pairs containing cytosines (no-CpG) in their sequence. (c) In MSP, primer pairs are designed to specifically amplify either methylated (M) or unmethylated (U) DNA by containing CpG site in their sequence that makes possible to distinguish the methylated sequence from the unmethylated sequence.