Journal of Taiwan Agricultural Research

Author:Yu-Chia Hsu, Hsin-Mei Ku and Chang-Sheng Wang*

Abstract:

    In genome, the DNA methylation levels are closely related with gene expression. Therefore, the analysis of DNA methylation has become an important field in molecular biology. Recently, the non-bisulfite and bisulfite techniques have been developed to detect the methylation of DNA. In the non-bisulfite treatment of DNA mehtylation analysis called methylation-sensitive amplified polymorphism (MSAP) which combines the amplified fragment length polymorphism (AFLP) technique with the application of restriction enzymes that are sensitive to methylation on the recognition sequences. The method is based on the utilization of isoschizomer enzymes that differ in their sensitivities to methylation to determine whether the methylation pattern with is full methylation, hemi-methylation or unmehtylation on specific DNA sequences. The MSAP method is mainly limited when the cytosine present on isoschizomer site, because it can not detect a number of cytosine methylation on DNA sequence and results in underestimation of DNA methylation. In the bisulfite treatment of DNA mehtylation detection, the methylation-specific PCR (MSP) technique is generally applied to study the methylation of CpG island. Primer design is a crucial step to the MSP technique, making this technique very sensitivity, allowing detecting small amounts of DNA methylation in the sample. MSP can also be applied to analyze large numbers of paraffin-embedded and microdissected samples. The methylation-sensitive single nucleotide primer extension (Ms-SNuPE) technique allows us to detect DNA methylation by using single nucleotide primer applying to the genomic DNA treated with sodium bisulfite to amplify and estimate the difference of methylation on specific CpG sites. With this method, DNA methylation can be quantified without using restriction enzymes, and the methylation at multiple CpG sites can be detected in a single reaction by using a multiplex oligonucleotide strategy. Low sensitivity is the mainly drawback of Ms-SNuPE technique. The recent developed COBRA (combined bisulfite restriction analysis) technique is a quantitative DNA methylation method. It can detect the methylation levels of specific locus in small amounts of genomic DNA, and accurate quantitative analysis on DNA methylation sequence. Furthermore, it can also apply to analyze a large amount of samples and paraffin-embedded tissue samples. Therefore, the application of COBRA technique in DNA methylation analysis has arose great attention.

Key words:Methylation, CpG island, Non-bisulfite, Bisulfite

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