text: This alternative method of methylation analysis also uses bisulfite-treated DNA but avoids the need to sequence the area of interest. Instead, primer pairs are designed themselves to be "methylated-specific" by including sequences complementing only unconverted 5-methylcytosines, or, on the converse, "unmethylated-specific", complementing thymines converted from unmethylated cytosines.  Methylation is determined by the ability of the specific primer to achieve amplification. This method is particularly useful to interrogate CpG islands with possibly high methylation density, as increased numbers of CpG pairs in the primer increase the specificity of the assay.  Placing the CpG pair at the 3'-end of the primer also improves the sensitivity.  The initial report using MSP described sufficient sensitivity to detect methylation of 0.1% of alleles.  In general, MSP and its related protocols are considered to be the most sensitive when interrogating the methylation status at a specific locus.
  The MethyLight method is based on MSP, but provides a quantitative analysis using quantitative PCR.  Methylated-specific primers are used, and a methylated-specific fluorescence reporter probe is also used that anneals to the amplified region.  In alternative fashion, the primers or probe can be designed without methylation specificity if discrimination is needed between the CpG pairs within the involved sequences. Quantitation is made in reference to a methylated reference DNA.  A modification to this protocol to increase the specificity of the PCR for successfully bisulfite-converted DNA (ConLight-MSP) uses an additional probe to bisulfite-unconverted DNA to quantify this non-specific amplification.
  Further methodology using MSP-amplified DNA analyzes the products using melting curve analysis (Mc-MSP).  This method amplifies bisulfite-converted DNA with both methylated-specific and unmethylated-specific primers, and determines the quantitative ratio of the two products by comparing the differential peaks generated in a melting curve analysis. A high-resolution melting analysis method that uses both quantitative PCR and melting analysis has been introduced, in particular, for sensitive detection of low-level methylation