unmethylated cytosine uracil are added to the chamber along with amplification reagents a polymerase enzyme and an excess of dntps the sample is thermocycled to perform pcr and the electrical output signal of the isfet is monitored as the thermocycling proceeds hydrogen ions are released during the incorporation of multiple dntps at the 3 u2032 end of the probe during the chain extension phase of pcr in the presence of a target sequence complimentary to the probe chain extension and hydrogen ion release will occur resulting in discrete fluctuations in the electrical output signal of the isfet this may be compared with the absence of a target sequence complimentary to the probe however since the amplification mixture will buffer the release of hydrogen ions amplification must proceed beyond a threshold number of cycles for buffering capacity of the sample to be overcome in order to generate an electrical output signal in response to a change in ph arising from chain extension during amplification in the presence of target dna any of the above embodiments may combine steps or introduce reagents in a different order the time at which the fluctuations occur and the magnitude of the fluctuations is monitored to allow sequencing of dna which in turn determines the location of methylated or.