利用酶联免疫反应鉴定芜菁SP11基因甲基化率的新方法

DNA甲基化是调控基因表达的一个重要方式,是表观遗传学研究的重点内容。目前测定某一基因DNA甲基化程度主要是先通过重亚硫酸盐转化,PCR扩增,再通过克隆、测序,检测PCR产物中胞嘧啶(C)转化为胸腺嘧啶(T)的比例。基于这种原理,建立了酶联免疫反应检测PCR产物中胞嘧啶转化为胸腺嘧啶的比例,计算出DNA甲基化率的新方法,并成功利用该方法测定了芜青(Brassica rapa L.ssp.rapiferu Metzg)纯合子和杂合子植株中隐性SP11基因启动子区域DNA甲基化程度。与原有克隆测序方法相比,该方法更加经济、省时。

Detecting SP11 Genes DNA Methylation Rate of Brassica rapa by PCR-ELISA

DNA methylation is an important way to regulate gene expression,which is a main content of epigenetic research field.The way to determinating DNA methylation rate of a gene is mainly by bisulfite conversion,then amplifying the converted DNA sequence by PCR,and cloning and sequencing the PCR production to measure the rate of cytosine converted into the thymine in the sequence.Based on this method,we developed anenzyme linked immunosorbent assays (ELISA) instead of cloning and sequencing to measure the rate of cytosine converted into the thymine in DNA sequence.We used this improved method to detecting the methylation rate of promotor region sequence of recessive SP11 in the heterozygote plants containing one dominate SP11 and one recessive SP11 of Brassica rapa L.ssp.rapiferu Metzg.Compared with the original method,the method reported here is more economical and time saving.