International Journal of Molecular Medical Science, 2024, Vol.14, No.4, 227-238 http://medscipublisher.com/index.php/ijmms 232 excellent quantitative accuracy and single-base pair resolution (Lutsik et al., 2011; Wreczycka et al., 2017). Specialized software tools, such as BiQ Analyzer HT, facilitate the analysis and visualization of high-throughput bisulfite sequencing data, making it more efficient and user-friendly (Lutsik et al., 2011). 6.2 Methylation-Specific PCR Methylation-specific PCR (MSP) is a technique used to detect methylation at specific CpG sites. This method involves bisulfite treatment of DNA followed by PCR amplification using primers specific for either methylated or unmethylated DNA. MSP is particularly useful for analyzing promoter regions of genes associated with cancer and other diseases. However, it has some limitations, such as potential biases in amplification and the need for careful primer design to avoid non-specific binding (How-Kit et al., 2015). Despite these drawbacks, MSP remains a widely used method due to its simplicity and specificity. 6.3 Pyrosequencing Pyrosequencing is a sequencing-by-synthesis method that quantitatively monitors the incorporation of nucleotides in real-time. This technique is highly effective for analyzing DNA methylation patterns, providing accurate and reproducible measures of methylation at multiple CpG sites within a sequence. Pyrosequencing can be used in conjunction with bisulfite treatment and PCR to quantify methylation levels at specific loci. It is particularly useful for studying promoter regions of genes and can be applied to both CpG and non-CpG methylation analysis. The method is also adaptable for high-throughput analysis, allowing the simultaneous examination of multiple samples (How-Kit et al., 2015). 6.4 High-Throughput methylation arrays High-throughput methylation arrays are designed to analyze DNA methylation across the genome at a large scale. These arrays use probes that hybridize to specific CpG sites, allowing for the simultaneous measurement of methylation levels at thousands of loci. This method is less labor-intensive and requires less DNA compared to traditional methods, making it suitable for large-scale studies. High-throughput methylation arrays are particularly useful for identifying genome-wide methylation changes and can serve as surrogate markers for global methylation levels. 6.5 Emerging Technologies Emerging technologies in DNA methylation analysis include single-cell methylome analysis, which allows for the examination of methylation patterns at the single-cell level. This technique is crucial for understanding cellular heterogeneity and the role of DNA methylation in cell differentiation and disease. Limiting dilution bisulfite pyrosequencing (LD-BSP) is one such method that enables methylation analysis of individual DNA molecules from a single or a few cells. This approach is particularly useful for studying rare alleles and small cell populations, providing insights into the methylation status of specific genes in individual cells (Hajj et al., 2015). 7 Clinical Implications of DNA Methylation in Colon Cancer 7.1 Biomarker Discovery for Early Detection DNA methylation has emerged as a promising biomarker for the early detection of colon cancer. The stability and frequency of DNA methylation changes make them ideal candidates for noninvasive biomarkers, detectable in bodily fluids such as blood and stool. For instance, methylation of specific gene promoters has been shown to be an early event in colorectal cancer (CRC) pathogenesis, providing a window for early diagnosis (Leygo et al., 2017; Jung et al, 2020; Wu et al., 2020). Studies have identified numerous differentially methylated regions (DMRs) that can distinguish between normal, adenoma, and carcinoma tissues, highlighting their potential in early cancer detection (Noguer et. al., 2023). Commercially available DNA methylation-based assays have already been integrated into clinical practice, underscoring their utility in early diagnosis (Davalos and Esteller, 2022). 7.2 Prognostic and Predictive Biomarkers DNA methylation patterns also serve as valuable prognostic and predictive biomarkers in colon cancer. Aberrantly methylated genes can provide insights into the likely course of the disease and patient outcomes. For example, the methylation status of genes such as GZMB has been associated with patient prognosis, offering a more accurate
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