Cancer Genetics and Epigenetics 2024, Vol.12, No.2, 88-96 http://medscipublisher.com/index.php/cge 89 unchanged. The treated DNA is then sequenced to determine the methylation status of cytosines. Bisulfite sequencing is considered the "gold standard" for DNA methylation analysis due to its high sensitivity and specificity (Reed et al., 2010). It has been effectively used in various studies, including the detection of methylation patterns in circulating cell-free DNA (cfDNA) for early cancer detection (Li et al., 2016; Liu et al., 2020). Whole-genome bisulfite sequencing (WGBS) has also been employed to study methylation heterogeneity in metastatic breast cancer, providing insights into intra-tumor heterogeneity (Luo et al., 2023). 2.2 Methylation-Specific PCR (MSP) Methylation-Specific PCR (MSP) is a PCR-based technique that allows for the rapid assessment of the methylation status of specific CpG sites. This method involves the initial modification of DNA by sodium bisulfite, followed by PCR amplification using primers specific for either methylated or unmethylated DNA. MSP is highly sensitive, capable of detecting as low as 0.1% methylated alleles, and can be performed on small quantities of DNA, including those extracted from paraffin-embedded samples. Variants of MSP, such as SMART-MSP, incorporate high-resolution melting (HRM) analysis to provide quantitative methylation detection and to distinguish between homogeneous and heterogeneous methylation (Kristensen et al., 2007). 2.3 Pyrosequencing Pyrosequencing is another method used to analyze DNA methylation. It involves sequencing by synthesis, where the incorporation of nucleotides is detected in real-time by the release of pyrophosphate. This method is advantageous for its ability to provide quantitative data on methylation levels at specific CpG sites. Pyrosequencing has been compared with bisulfite sequencing PCR (BSP) and found to be equally effective in detecting hypomethylation and mixed methylation, although BSP may be more sensitive for detecting strong hypermethylation (Kristensen and Hansen, 2019). Massively parallel bisulfite pyrosequencing has been used to reveal the molecular complexity of breast cancer-associated cytosine-methylation patterns in both tissue and serum DNA (Korshunova et al., 2007). 2.4 Microarrays Microarrays are used for high-throughput analysis of DNA methylation across the genome. This technology involves hybridizing bisulfite-treated DNA to probes on a microarray chip, allowing for the detection of methylation at thousands of CpG sites simultaneously. Microarrays have been employed to evaluate the methylation levels of candidate genes in plasma cfDNA for breast cancer early detection, demonstrating the feasibility of using this method for non-invasive cancer diagnostics (Li et al., 2016). 2.5 Comparative analysis with methods Each of these technologies has its own strengths and limitations. Bisulfite sequencing is highly sensitive and specific but can be labor-intensive and costly, especially for whole-genome applications (Reed et al., 2010; Luo et al., 20023). MSP is rapid and highly sensitive but is limited to the analysis of specific CpG sites and may produce false positives if not carefully controlled (Giridhar et al., 2023). Pyrosequencing provides quantitative data and is less labor-intensive than bisulfite sequencing but may be less sensitive for detecting strong hypermethylation. Microarrays offer high-throughput capabilities but may lack the single-base resolution provided by sequencing methods. 3 Advances in Understanding DNA Methylation in Breast Cancer 3.1 Methylation patterns in breast cancer Recent studies have significantly advanced our understanding of DNA methylation patterns in breast cancer. Massively parallel bisulphite pyrosequencing has revealed the molecular complexity of cytosine-methylation patterns in both tissue and serum DNA from breast cancer patients. This comprehensive analysis demonstrated that tumor samples exhibit more variation in methylation levels compared to normal samples, highlighting the potential of these patterns as biomarkers for early detection (Korshunova et al., 2007). Additionally, genome-wide DNA methylation profiling has identified numerous hypermethylated loci/genes in breast tumors, which are associated with clinical features such as estrogen receptor and progesterone receptor status, tumor relapse, and lymph node metastasis (Hill et al., 2011). High-throughput MALDI-TOF mass array analysis has further identified
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