Exploring CpG Sites by Sequencing Technology

What is a CpG island?

CpG islands are specialized regions within the genome, frequently enriched with dinucleotide "CGs," notably found in the promoter regions of many genes, especially those associated with housekeeping functions. These CpG sites play a crucial role in the recruitment of transcription factors, influencing gene expression. The heightened methylation status of CpG islands further modulates the recruitment of transcription factors, thereby impacting gene regulation. In the context of mammalian DNA methylation, CG-type methylation prevails, and CpG sites, abundant in CpG dinucleotides, are commonly situated proximal to transcriptional regulatory regions. Remarkably, these CpG islands are linked to approximately 56% of the coding genes in the human genome, underscoring the paramount importance of studying their methylation status in the realm of methylation research.

CpG density, closely intertwined with DNA methylation dynamics in various tissues, categorizes the genome into five regions: CpG island (CGI), the area within 2kb downstream of the CpG island (CpG shore), and the zone within 2kb both upstream and downstream of the CpG island shore (CpG shelve). The methylation levels of each region are meticulously assessed individually.

Applications of CpG Research

• Epigenetic Research: DNA methylation serves a crucial role in preserving normal cellular function, maintaining chromosome structure, and facilitating X-chromosome inactivation. Exploring CpG islands contributes to the advancement of human epigenetics research.
• Early Cancer Detection: Elevated methylation levels in gene promoters are frequently observed in the transcription of genes associated with malignant tumors. This phenomenon is considered a hallmark of cellular malignant transformation, enabling the early detection of cancer through gene methylation sequencing.
• Embryonic Development Research: DNA methylation plays a pivotal role in genetic imprinting and embryonic development. CpG island studies offer valuable insights into understanding the intricate processes involved in embryonic development.
• iPSC and Stem Cell Research: CpG islands are instrumental in the analysis of methylation patterns, providing insights into the differentiation processes of stem cells and induced pluripotent stem cells (iPSCs).

Bisulfite Sequencing in CpG Research

BS-Seq (Bisulfite Sequencing) stands out as the preeminent method for comprehensive genome-wide methylation detection. This technique involves the conversion of unmethylated cytosines (C) to uracil (U) through bisulfite treatment. By integrating second-generation sequencing and subsequent analysis, BS-Seq enables high-throughput detection of methylation sites.

In the bisulfite treatment process, unmethylated cytosines undergo conversion to uracil, while methylated cytosines remain unaltered. PCR primers are meticulously designed based on the sequence of the target site or region of interest. The amplified products are subjected to various detection methods, including PCR electrophoresis, sequencing, fluorescence quantification, and next-generation sequencing (NGS). The obtained results are then analyzed to identify methylated sites and determine the frequency of methylation.

The procedural steps of the BSP method are outlined as follows:

• BSP Primer Design: Design BSP primers tailored to the target gene region, typically yielding an amplification product of around 300 base pairs.
• Bisulfite Treatment of Sample Genome: Treat the genomic DNA with bisulfite to induce the conversion of unmethylated cytosines to uracil.
• PCR Amplification: Employ PCR to amplify the region of interest within the sample.
• Cloning into Vector: Purify the PCR products and ligate them into commonly used vectors.
• Plasmid Extraction and Sequencing: Extract and sequence plasmids from a selection of positive clones, typically selecting 10 for inoculation and culture. This step allows for the detailed examination of methylation patterns at the molecular level.

Nanopore Sequencing

The ONT (Oxford Nanopore Technologies) platform distinguishes the methylation status of bases, such as 5mC or 6mA, by analyzing the alterations in the electrical signal as the base traverses the nanopore microwell.

Nanopore sequencing exhibits the remarkable capability to directly identify methylated cytosines, including those at CpG sites, eliminating the necessity for bisulfite conversion. This technology is particularly adept at discerning methylation patterns in high-density clusters known as CpG islands (CGIs).