Detection of DNA Methylation in Genomic DNA by UHPLC-MS/MS.
Detection of DNA Methylation in Genomic DNA by UHPLC-MS/MS.
复制标题
DOI：
10.1007/978-1-0716-0876-0_7
复制
发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Greer EL
中科院分区：
文献类型：
--
作者：
Boulias K;Greer EL
DNA甲基化可以作为一种定向表观遗传信号修饰或对DNA损伤的反应来标记DNA。随着时间的推移，检测DNA甲基化的方法变得越来越具体和敏感。检测DNA甲基化的常规方法；从纸层析，到差分限制性内切酶消化偏好，再到点印迹，最近被超高高效液相色谱-质谱联用（UHPLC-MS/MS）所补充，以准确定量特定的DNA甲基化。甲基化DNA也可以通过甲基化DNA免疫沉淀测序（MeDIP-seq）或单分子实时测序（SMRTseq）进行测序，以确定DNA甲基化的基因组位置。本文描述了一种采用三重四极杆液相色谱-串联质谱（QQQ-LC-MS/MS）检测和定量基因组DNA中表观遗传信号DNA甲基化修饰的方法，包括n6 -甲基腺嘌呤（6mA）、n4 -甲基胞嘧啶（4mC）和c5 -甲基胞嘧啶（5mC）。UHPLC-MS/MS方法的高灵敏度和使用纯核苷的校准标准允许DNA甲基化的准确定量。
DNA methylation serves to mark DNA as either a directed epigenetic signaling modification or in response to DNA lesions. Methods for detecting DNA methylation have become increasingly more specific and sensitive over time. Conventional methods for detecting DNA methylation; ranging from paper chromatography, to differential restriction enzyme digestion preference, to dot blots, have more recently been supplemented by ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS/MS) to accurately quantify specific DNA methylation. Methylated DNA can also be sequenced by either methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq) or single-molecule real-time sequencing (SMRTseq) for identifying genomic locations of DNA methylation. Here we describe a protocol for the detection and quantification of epigenetic signaling DNA methylation modifications including, N6-methyladenine (6mA), N4-methylcytosine (4mC) and C5-methylcytosine (5mC) in genomic DNA by Triple Quadrupole Liquid Chromatography coupled with tandem Mass Spectrometry (QQQ-LC-MS/MS). The high sensitivity of the UHPLC-MS/MS methodology and the use of calibration standards of pure nucleosides allow the accurate quantification of DNA methylation.