利用抗5-甲基胞嘧啶单克隆抗体检测人类重复DNA亚群中的高度甲基化

Detection of heavy methylation in human repetitive DNA subsets by a monoclonal antibody against 5-methylcytosine.

作者信息

Sano H, Imokawa M, Sager R

机构信息

Biotechnology Institute, Akita Prefectural College of Agriculture, Japan.

出版信息

Biochim Biophys Acta. 1988 Nov 10;951(1):157-65. doi: 10.1016/0167-4781(88)90036-x.

DOI:10.1016/0167-4781(88)90036-x

PMID:2847796

Abstract

A monoclonal antibody (IgM) against 5-methylcytosine (mC) was isolated and characterized. It showed a high specificity for mC with a cross-reactivity of less than 1% with cytosine and 0.1% with thymidine. An improved immunohybridization method, originally developed with polyclonal antibodies (Sano et al. (1980) Proc. Natl. Acad. Sci. USA 77, 3581), was applied to detect mC in immobilized DNA using the new monoclonal preparation. Human genomic DNA was cleaved with the restriction enzyme EcoRI and successively fractionated by malachite-green affinity chromatography and agarose gel electrophoresis. The fractionated DNA was transferred to nitrocellulose paper and treated with the anti-mC monoclonal antibody. Heavy methylation was observed in EcoRI-ladders of repetitive sequences of 1360, 1750, 2200 and 3400 bp, while 340, 660 and 2700 bp fragments were less methylated. The results show that methylation occurs in limited subsets of satellite II and III repetitive DNAs that contain high amounts of methylatable CpG dinucleotides, or CpG clusters.

摘要

一种针对5-甲基胞嘧啶（mC）的单克隆抗体（IgM）被分离并进行了表征。它对mC表现出高度特异性，与胞嘧啶的交叉反应性小于1%，与胸腺嘧啶的交叉反应性为0.1%。一种最初用多克隆抗体制备的改进免疫杂交方法（Sano等人（1980年），《美国国家科学院院刊》77卷，3581页），被应用于使用新的单克隆制剂检测固定化DNA中的mC。人类基因组DNA用限制性内切酶EcoRI切割，然后依次通过孔雀石绿亲和色谱和琼脂糖凝胶电泳进行分级分离。分级分离后的DNA转移到硝酸纤维素纸上，并用抗mC单克隆抗体处理。在1360、1750、2200和3400 bp重复序列的EcoRI梯状条带中观察到高度甲基化，而340、660和2700 bp片段的甲基化程度较低。结果表明，甲基化发生在卫星II和III重复DNA的有限子集中，这些子集含有大量可甲基化的CpG二核苷酸或CpG簇。