**PCR conditions:**

PCR buffer 1×, MgCl: 2 2.5 mM, DMSO: 5% (v/v), dNTP: 12.5 mM, Primer Forward: 10 pmol, Primer Reverse: 10 pmol, Taq Polymerase: 1 U (5 U/μL), Template DNA: 100 ng and dH2O were used to obtain a total of 50 μL, and PCR thermal cycling procedure was as follows:

This investigation allows determining whether the region of interest is methylated and quantifying methylation by measuring the band intensity with any gel analysis system. If desired, results 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).

#### **5.2. Evaluation of agarose gel imaging**

The resulting primers were stained with ethidium bromide on 2% agarose gel, and agarose gel findings were evaluated by examination under ultraviolet light (**Figures 7–10**).

**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 is observed in other samples [32].

Example run:

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

Methylation-specific polymerase chain reaction: DNA purity was measured at wavelengths of 260 and 280 nm on spectrophotometry, and DNA quantification was performed using the DNA (μg/mL) = A260 × Dilution Factor × 50 (coefficient) formula at 260 nm UV. Subsequently, 10 μL of DNA was taken from each sample and bisulphite modification was carried out for DNA with Millipore CpGenome modification kit according to the manufacturer's instructions. This modification converts the cytosines of the unmethylated region to thymines. For the region thought to be altered in this manner, CpG sequences in exon 1 of the prestin gene were detected using the MethPrimer V1.1 beta program [30]. MSP was conducted according to the following protocols to investigate methylation utilising the PCR primers for exon 1 of the prestin gene stated below, and PCR conditions for the methylated and unmethylated regions are as follows:

PCR buffer 1×, MgCl: 2 2.5 mM, DMSO: 5% (v/v), dNTP: 12.5 mM, Primer Forward: 10 pmol, Primer Reverse: 10 pmol, Taq Polymerase: 1 U (5 U/μL), Template DNA: 100 ng and dH2O were used to obtain a total of 50 μL, and PCR thermal cycling procedure was as follows:

This investigation allows determining whether the region of interest is methylated and quantifying methylation by measuring the band intensity with any gel analysis system. If desired, results

then analysed by visualisation with ethidium bromide agarose gel imaging systems.

**5.1. Sample reaction: (for the prestin gene promoter region in guinea pigs)**

**Methylated region (M: methylated):**

PCR product: 164 base pairs (bp).

PCR product: 163 bp.

**PCR conditions:**

12 Chromatin and Epigenetics

M-Forward: ATGTTGAAGAAAATGAAATTTTCGT,

UnM-Reverse: AAACTACCAAACAAAAAACAACATC,

M-Reverse: ACTTATCCCCGATAAAATCCG,

**Unmethylated region (UnM: unmethylated):** UnM-Forward: TTTATTTTTAGAAGGTTGTGG,

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

#### **5.3. MS RFLP**

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


and cut only specific methylated regions. The most commonly used restriction enzymes for this purpose are HpaII and MspI.

This process digests the methylated CpG (if present) in the DNA region of interest. If that region is not methylated, digestion occurs via HpaII, an isomer of MspI. Concurrent use of these two enzymes provides insight on the methylation status of the region in question.
