text: MS-SnuPE employs the primer extension method initially designed for analyzing single-nucleotide polymorphisms.  DNA is bisulfite-converted, and bisulfite-specific primers are annealed to the sequence up to the base pair immediately before the CpG of interest. The primer is allowed to extend one base pair into the C (or T) using DNA polymerase terminating dideoxynucleotides, and the ratio of C to T is determined quantitatively.
  A number of methods can be used to determine this C:T ratio. At the beginning, MS-SnuPE relied on radioactive ddNTPs as the reporter of the primer extension.  Fluorescence-based methods or Pyrosequencing can also be used. However, matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry analysis to differentiate between the two polymorphic primer extension products can be used, in essence, based on the GOOD assay designed for SNP genotyping.  Ion pair reverse-phase high-performance liquid chromatography (IP-RP-HPLC) has also been used to distinguish primer extension products.