Comparative isoschizomer profiling of cytosine methylation: The HELP assay
Comparative isoschizomer profiling of cytosine methylation: The HELP assay
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DOI：
10.1101/gr.5273806
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发表时间：
2006-08-01
期刊：
影响因子：
7
通讯作者：
Greally, John M.
中科院分区：
文献类型：
--
作者：
Khulan, Batbayar;Thompson, Reid F.;Greally, John M.
利用一种甲基化敏感的限制性内切酶及其甲基化不敏感的同分异构体的基因组代表共杂交，对小鼠基因组中6.2 Mb的胞嘧啶甲基化分布进行了检测。这种被称为HELP （HpaII微小片段富集通过连接介导的PCR）的分析，允许胞嘧啶甲基化的基因组内分析和基因组间比较。基因组内图谱显示，大部分基因组是连续的甲基化序列，偶有低甲基化位点簇，通常但不限于启动子和CpG岛。基因组间比较发现，生精细胞和脑细胞之间胞嘧啶甲基化存在显著差异，确定了223个新的候选组织特异性差异甲基化区域（T-DMRs）。亚硫酸氢盐焦磷酸测序证实了这四个候选基因是T-DMRs，而定量RT-PCR对两个启动子上有T-DMRs的基因进行了定量RT-PCR，结果显示HELP数据与这些位点上的基因活性相关。HELP检测稳健、定量、准确，为基因组中胞嘧啶甲基化的分布和动态性质提供了新的见解。
The distribution of cytosine methylation in 6.2 Mb of the mouse genome was tested using cohybridization of genomic representations from a methylation-sensitive restriction enzyme and its methylation-insensitive isoschizomer. This assay, termed HELP ( HpaII tiny fragment Enrichment by Ligation-mediated PCR), allows both intragenomic profiling and intergenomic comparisons of cytosine methylation. The intragenomic profile shows most of the genome to be contiguous methylated sequence with occasional clusters of hypomethylated loci, usually but not exclusively at promoters and CpG islands. Intergenomic comparison found marked differences in cytosine methylation between spermatogenic and brain cells, identifying 223 new candidate tissue-specific differentially methylated regions (T-DMRs). Bisulfite pyrosequencing confirmed the four candidates tested to be T-DMRs, while quantitative RT-PCR for two genes with T-DMRs located at their promoters showed the HELP data to be correlated with gene activity at these loci. The HELP assay is robust, quantitative, and accurate and is providing new insights into the distribution and dynamic nature of cytosine methylation in the genome.