Proof of principle of novel technique of methylation analysis

In this work we demonstrate a novel method of methylation detection that utilises immunoaffinity to detect the presence of methylated DNA hybridised to a cDNA microarray. We use a monoclonal antibody specific to 5 methylcytosine to detect the presence of 5 methylcytosine in genomic DNA from human fibroblasts bearing the karyotype 45 XO. We report that over 2900 genes show the presence of methylation in this condition. We also report that 165 genes are consistently methylated in all replicates of these experiments. The methylated genes show a uniform distribution over all the chromosomes. The gene ontology of these also indicates no functional correlation between the genes that are methylated. We detect the presence of methylation in IGF2, an imprinted gene and thus known to harbour DNA methylation. The method is extremely specific and offers a quick and efficient way to analyse the methylation landscape on a high throughput scale. This method uses existing technology to assess methylation and thus can integrate very efficiently into any platform used. A method that detects DNA methylation that is not restricted to specific sequences would provide a useful platform to analyse methylation distribution among the genes in any given phenotypic condition.Availability of additional approaches to examine DNA methylation would lead to increase in our understanding of the methylome and would help define the intrinsic and extrinsic factors which govern it. Microarray based high throughput methods have been extensively used for gene expression analysis. The method described uses a microarray to analyse DNA methylation levels in a gene/cDNA/oligo specific manner. Here, genomic DNA is fragmented and used for hybridization to microarray slides. The presence of DNA cytosine methylation in the hybridized DNA is detected by employing Cy3 labelled anti 5mC( 5-methyl cytidine) antibody (monoclonal). The resolution provided by this method is dependent upon the microarray platform used. This method can be adapted to any platform thus enabling seamless integration with existing technology of gene expression analysis The experiment involves the use of genomic DNA from human fibroblasts with the karyotype 45XO and 47XXX. This experiment was performed to analyse the methylation distribution in the human genome in the absence of X chromosome inactivation and this was compared to the methylation distribution in the genome in the background of XCI. 3 technical replicates were used for the XO sample and 2 for XXX. The methylation was estimated by calculating the fold florescence intensity to control spots for the single channel experiments carried out. Each replicate was normalised individually with the background florescence. The normalised florescence values were used for further comparison between and within replicates.

本研究展示了一种全新的甲基化检测方法，该方法借助免疫亲和技术，对与互补DNA（cDNA）微阵列杂交的甲基化DNA进行检测。我们使用针对5-甲基胞嘧啶（5-methylcytosine）的单克隆抗体，对核型为45,XO的人类成纤维细胞的基因组DNA中的5-甲基胞嘧啶进行检测。本研究发现，在该核型条件下，超过2900个基因存在甲基化现象；同时有165个基因在所有实验重复中均呈现稳定的甲基化状态。甲基化基因在所有染色体上呈均匀分布，且这些基因的基因本体论分析结果显示，甲基化基因之间不存在功能相关性。我们在印记基因IGF2中检测到了甲基化，而该基因因属于印记基因，其携带DNA甲基化已是已知结论。该方法特异性极强，可提供一种快速高效的高通量方式分析基因组甲基化图谱。本方法依托现有技术开展甲基化评估，因此能够高效整合至各类现有实验平台。

一种无需依赖特定序列即可检测DNA甲基化的方法，将为分析任意特定表型条件下的基因甲基化分布提供实用平台。更多可用于检测DNA甲基化的方法，将有助于加深我们对甲基化组（methylome）的理解，并助力阐明调控甲基化组的内在与外在因素。基于微阵列的高通量方法已被广泛应用于基因表达分析。本研究所述方法借助微阵列，以基因/互补DNA/寡核苷酸特异性的方式分析DNA甲基化水平。具体而言，首先将基因组DNA片段化，随后用于与微阵列玻片杂交；杂交后的DNA中胞嘧啶甲基化的检测，通过使用Cy3标记的抗5-甲基胞苷（5-methyl cytidine）单克隆抗体完成。

该方法的分辨率取决于所使用的微阵列平台，且可适配任意平台，因此能够与现有的基因表达分析技术实现无缝整合。本实验使用核型为45,XO与47,XXX的人类成纤维细胞基因组DNA，旨在分析不存在X染色体失活（X chromosome inactivation, XCI）时的人类基因组甲基化分布，并将其与存在X染色体失活背景下的基因组甲基化分布进行对比。其中，XO样本设置3次技术重复，XXX样本设置2次技术重复。

甲基化水平通过计算单通道实验中各样本与对照点的荧光强度倍数进行估算。每个重复样本均单独以背景荧光进行归一化处理，归一化后的荧光值用于重复间及重复内的后续比较。