DNA甲基化分析技術之發展與應用

Progress and Application of DNA Methylation Detection

許育嘉(Yu-Chia Hsu) ； 古新梅(Hsin-Mei Ku) ； 王強生(Chang-Sheng Wang)

《台灣農業研究》 54卷2期 (2005/06) Pp. 71-82

https://doi.org/10.6156/JTAR/2005.05402.01

摘要

基因組中DNA甲基化程度與基因表現密切相關，因此，逐漸受到重視。目前有兩類技術運用於DNA甲基化程度的研究，包括非重亞硫酸鹽(non-bisulfite)和重亞硫酸鹽(bisulfite)的方法。在不使用重亞硫酸鹽處理之DNA甲基化分析，是結合AFLP的技術，配合對甲基化敏感的限制酵素之使用，稱甲基敏感擴增多型性方法(MSAP)，此方法乃是利用酵素對於不同甲基化程度辨識的差異，判斷特定DNA序列之甲基化形式，為完全甲基化或是未甲基化，此方法主要受限於只能研究在同辨識位中所出現的胞嘧啶，不能測定到某些胞嘧啶受甲基化的情形，會導致低估DNA甲基化的程度。在使用重亞硫酸鹽分析DNA甲基化方面，以甲基特異PCR方法(MSP)為最廣泛應用於CpG島甲基化的研究，此方法成功的關鍵在於引子的設計，由於此法具有高靈敏度，所以能允許偵測微量樣品DNA甲基化的情形，甚至包括由埋蠟或是顯微解剖來的組織均可分析。甲基化敏感的單核酸引子延長反應法(Ms-SNuPE)是藉由單一核苷酸引子，在處理過重亞硫酸鹽的DNA上進行擴增，可以評估特定CpG島甲基化的不同，此法能進行定量分析，不需利用限制酵素，能藉由利用多個引子的策略，在引子擴增反應下，分析多個CpG位置，由於需仰賴放射性強度的測定，靈敏度較差為此方法的主要缺點。組合重亞硫酸鹽限制分析法(COBRA)，為一個定量技術，它能測定小量基因組DNA中，特定基因座DNA甲基化的程度，由於此法能針對DNA甲基化的情形作準確的定量分析，並可大量分析樣品與應用在埋蠟切片組織的分析，所以在DNA甲基化分析的領域已受到重視。

關鍵字

甲基化 ； CpG島 ； 重亞硫酸鹽 ； 非重亞硫酸鹽

並列摘要

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.

並列關鍵字

Methylation ； CpG island ； Non-bisulfite ； Bisulfite