Monitoring of reaction dynamics by real-time PCR. A) Monitoring of the deamination reaction as a function of deamination time using primers designed for an unmethylated part of the MLH1 promoter. Three different primer sets were used. The first set (MLH1 UF and MLH1 R) binds to areas with no cytosines, and hence amplify both deaminated and undeaminated DNA. However, since deamination creates 2 uncomplementary DNA strands, only one strand of the deaminated DNA can function as a template. The second set (MLH1 DF and MLH1 R) cytosine has been replaced by thymine making it specific to deaminated DNA. The third set (MLH1 UDF and MLH1 UDR) contains cytosines making it specific to undeaminated DNA. Real-time PCR detection of the 3 products was performed with a common molecular beacon (MLH1 beacon). B) Binding sites of beacon and primers used are shaded. Top strand is the undeaminated DNA sequencing. Bottom strand is the deaminated sequence. A horizontal line between the two strands illustrates no difference in sequence, ":" marks the positions of cytosine converted to uracil during deamination, and "+" marks the positions of CpG dinucleotides. Primer sequences can be found in Table 1. The universal primers, MLH1 UF and MLH1 R, contain Inosine at one position each.
Pedersen et al. BMC Molecular Biology 2012 13:12 doi:10.1186/1471-2199-13-12