一、表观遗传学简介

       表观遗传学是研究基因的核苷酸序列不发生改变的情况下，基因表达的可遗传的变化的一门遗传学分支学科。DNA甲基化(DNAmethylation)、组蛋白修饰(histone modification)、基因组印记(genomic imprinting)、RNA编辑(RNA edi-ting)、基因沉默、核仁显性、休眠转录子激活和性别相关性基因剂量补偿效应等都是典型的表观遗传现象。

表观遗传调控研究技术分为转录前调控研究技术和转录后调控研究技术。转录前调控研究技术主要包括：MSP、BSP、ChIP、EMSA、DNA Pull down、酵母单杂交（Y1H）；转录后调控研究技术主要包括RIP、RNA Pull down 、双荧光素酶报告系统等。

金开瑞致力于ChIP、EMSA、DNA/RNA pulldown、RIP、双萤光素酶系统、MSP（甲基化特异性PCR法）、BSP(亚硫酸氢钠处理后测序法)、MeDIP-seq等实验技术服务。除了提供**的技术服务外，我们还可以为您提供表观遗传学整体项目设计，整体项目实施，整体项目跟踪服务，金开瑞多年的实验和项目管理经验，将助力您表观遗传研究。

整体研究框架

二、转录前调控研究技术

MSP(甲基化特异性PCR)
        甲基化特异性是指用亚硫酸氢钠处理基因组DNA，未甲基化的胞嘧啶变成尿嘧啶，而甲基化的胞嘧啶不变，然后用3对特异性的引物对所测基因的同一核苷酸序列进行扩增。扩增产物用DNA琼脂糖凝胶电泳，凝胶扫描观察分析结果。

技术流程

提取基因组DNA，并定量—亚硫酸氢钠处理DNA—DNA修饰纯化后回收—引物设计合成，PCR检测—琼脂糖凝胶电泳

技术运用

(A): Methylation specific PCR of CASP8, MASPIN, TMS1 and MDR1 in prostate cancer cells. DNA from prostate cancer cells were Bisulfite modified using EZ methylation kit (Zymo Research) and then PCR done using methylation specific primers and unmethylation specific primers. Because in the cells sometimes methylated and unmethylated both copies are present, so depending upon the number copies they get amplified. (B): Methylation specific PCR of CASP8, MASPIN, TMS1 and MDR1 in 5-aza-dC (5µM/48hrs) treated prostate cancer cells. (C) Bisulfite sequencing chromatogram of TMS1 gene showing methylation in LNCaP, DU145 and PC3, partial methylation in PC3 and Unmethylation in RWPE1. (B): Diagrammatic representation of methylation pattern of TMS1 and MDR1 gene at different promoter positions in RWPE1, LNCaP, DU145 and PC3. Dark circles (●) represents methylated sites, shadowed circles (An external file that holds a picture, illustration, etc.Object name is nihms159165ig1.jpg) represent partially methylated sites and blank circles (○) represents unmethylated sites in TMS1 and MDR1 promoter region.

(Mishra DK, Chen Z, Wu Y, Sarkissyan M, Koeffler HP, Vadgama JV. Global methylation pattern of genes in androgen-sensitive and androgen-independent prostate cancer cells. Mol Cancer Ther. 2010 Jan;9(1):33-45.)

亚硫酸氢盐处理后测序法（bisulfite sequencing PCR BSP）

       亚硫酸氢盐处理后测序法：这种方法一度被认为是DNA甲基化分析的金标准。它的过程如下：经过亚硫酸氢盐处理后，用PCR扩增目的片段，并对PCR产物进行测序，将序列与未经处理的序列进行比较，判断CpG位点是否发生甲基化。这种方法可靠，且**度高，能明确目的片段中每一个CpG位点的甲基化状态，但需要大量的克隆测序，过程较为繁琐、 昂贵。

技术流程
        基因组DNA的提取—亚硫酸氢钠修饰基因组DNA—引物设计及修饰后DNA用于PCR—PCR产物的凝胶回收—PCR产物与T载体的连接和转化、克隆筛选

技术运用

Rad23b and Ddit3 methylation determined by BSP. Bisulfite treated DNA was amplified by PCR with BSP primers. PCR products were cloned into the pUC57 vector, and five clones selected and sequenced from each sample. Methylation level was defined as the ratio of methylated CpG sites in all clones. (A) Typical sequencing results; (B) early effects, tissues were collected 2 h postirradiation; (C) delay effects, 1 month postirradiation. *P,0.05 versus control.

Wang J, Zhang Y, Xu K, Mao X, Xue L, Liu X, Yu H, Chen L, Chu X. Genome-wide screen of DNA methylation changes induced by low dose X-ray radiation in mice. PLoS One. 2014 Mar 10;9(3):e90804.）
 

MeDIP-seq（甲基化DNA免疫共沉淀测序）
MeDIP-Seq（Methylated DNA Immunoprecipitation Sequencing）测序是基于抗体富集原理进行测序的全基因组甲基化检测技术，采用甲基化DNA免疫共沉淀技术，通过5'-甲基胞嘧啶抗体特异性富集 基因组上发生甲基化的DNA片段，然后通过高通量测序可以在全基因组水平上进行高精度的CpG密集的高甲基化区域研究。
技术流程

MeDIP流程图

技术运用

Illustration of dA6m DNA immunoprecipitation.

Genomic DNA is isolated and any RNAs are eliminated by RNAse treatment. Next, the DNA is fragmented and enriched by DNA immunoprecipitation using an antibody against dA6m. The enriched fraction can then be analyzed by subsequent deep sequencing.

（Koziol MJ, Bradshaw CR, Allen GE, Costa AS, Frezza C. Identification of Methylated Deoxyadenosines in Genomic DNA by dA(6m) DNA Immunoprecipitation. Bio Protoc. 2016 Nov 5;6(21).）