Introduction of Some New Technologies (4)

November 9, 2018

Bisulfite Sequencing PCR, BSP

DNA methylation strongly affects chromatin structure and the regulation of gene expression. For many years, bisulfite sequencing PCR (BSP) has served as the “gold standard” for measuring DNA methylation. BSP has high reliability and accuracy, and can accurately determine the methylation status of each CpG site in the fragment.

Treatment of DNA with bisulfite converts cytosine residues to uracil but leaves 5-methylcytosine residues unaffected. The BSP primers were designed for PCR amplification of target regions. Sequencing of the amplified products can determine which CpG sites have been methylated; generally, the sequencing success rate of cloned PCR products will be higher.

Workflow of Bisulfite Sequencing PCR

• DNA Extraction
• Bisulfite Conversion
• Primer Design and PCR Amplification
• Sequencing
• Data Analysis

Sample Requirements

• Sample type: Genomic DNA.
• Starting amount of total DNA: ≥ 2 µg
• Sample conc.: ≥ 100 ng/µl
• Sample volume ≥20ul

Advantages of Bisulfite Sequencing PCR

1. Both qualitative and quantitative research can be done;
2. High sensitivity;
3. Primer design is relatively simple.

Methylation-specific PCR,MSP

This alternative method of methylation analysis also uses bisulfite-treated DNA but avoids the need to sequence the area of interest. Instead, primer pairs are designed themselves to be "methylated-specific" by including sequences complementing only unconverted 5-methylcytosines, or, on the converse, "unmethylated-specific", complementing thymines converted from unmethylated cytosines. Methylation is determined by the ability of the specific primer to achieve amplification. This method is particularly useful to interrogate CpG islands with possibly high methylation density, as increased numbers of CpG pairs in the primer increase the specificity of the assay. Placing the CpG pair at the 3'-end of the primer also improves the sensitivity. The initial report using MSP described sufficient sensitivity to detect methylation of 0.1% of alleles. In general, MSP and its related protocols are considered to be the most sensitive when interrogating the methylation status at a specific locus.

Workflow of Methylation-specific PCR

• DNA Extraction
• Bisulfite Conversion
• DNA purification and recovery
• PCR
• Electrophoresis

Sample Requirements

• Sample type: Genomic DNA.
• Starting amount of total DNA: ≥ 2 µg
• Sample conc.: ≥ 100 ng/µl
• Sample volume ≥20ul

Advantages of Methylation-specific PCR

1. Avoid using restriction enzyme and subsequent related issues;
2. High sensitivity.