**5. Methylation-specific PCR (MSP)**

MSP is an established and the most commonly utilised method to determine the presence or absence of methylation in a gene region of interest as well as the extent of methylation, if any [30, 31].

In this method, DNA is initially subjected to total bisulphite treatment. In this way, all of the unmethylated cytosines in the DNA sequence are transformed into thymine. However, the methylated cytosines remain unchanged (**Figure 5**). This results in a motif change in the methylated region. Subsequently, spectrophotometric DNA quantification is conducted with DNA samples. For this, measurements are made at wavelengths of 260/280 nm and multiplied by the dilution factor to determine the amount of DNA as nanogram per microlitre; and according to these amounts, MS-PCR is performed taking care to include equal amounts of DNA samples in the PCR. In this type of PCR, the PCR is conducted in two separate tubes for each sample. While the primer specific for the methylated region is added into one tube, the other tube contains the primer for the unmethylated region, and PCR is performed as 35–40 cycles. PCR samples are then analysed by visualisation with ethidium bromide agarose gel imaging systems.

may also be obtained while real-time PCR is performed with SYBR-green or Taqman or similar fluorescent probes (the probe must be designed according to the bisulphite DNA sequence).

Logic of Epigenetics and Investigation of Potential Gene Regions

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The resulting primers were stained with ethidium bromide on 2% agarose gel, and agarose

**Figure 9.** Results of polymerase chain reaction in all study groups at the time point of second week for prestin (K1–K3: Group 1; K3–K6: Group 2; T1–T6: Group 3). Complete methylation is seen in sample 6T, while heterozygous methylation

Another method used to detect any DNA methylation is the methylation-specific restriction fragment length polymorphism (MS-RFLP) method, which produces methylation-specific digestion. This method employs restriction enzymes obtained from bacteria, which recognise

**Figure 10.** Imaging of MSP and un-MSP PCRs of survivin exon 1. Well 1: marker, 100 bp marker; well 2: positive control; 3–4, 5–6, 7–8, 9–10, 11–12, while unmethylated PCR results are found to be at the same (+) intensity in MSP and un-MSP sample wells of the same cases, the following methylated PCR results were not observed except the control DNA (+), and

5' C ↓ C G G 3' 3' G G C ↑ C 5'

all samples were accepted as unmethylated. Well 13 is the negative control of the reaction (**Figure 10**).

gel findings were evaluated by examination under ultraviolet light (**Figures 7–10**).

**5.2. Evaluation of agarose gel imaging**

Example run:

is observed in other samples [32].

**5.3. MS RFLP**
