Droplet Magnetofluidic Assay Platform for Quantitative Methylation-Specific PCR.
Droplet Magnetofluidic Assay Platform for Quantitative Methylation-Specific PCR.
复制标题
用于定量甲基化特异性 PCR 的液滴磁流控检测平台。
DOI：
10.1007/978-1-0716-1811-0_13
复制
发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Wang,Tza-Huei
中科院分区：
文献类型：
--
作者：
Stark,Alejandro;Trick,Alexander;Pisanic2nd,ThomasR;Wang,Tza-Huei
早期癌症检测需要识别由肿瘤发生引起的细胞变化。在肿瘤发展早期发生的异常DNA甲基化模式已被广泛鉴定为多种类型癌症肿瘤的早期生物标志物。甲基化特异性PCR（MSP）允许在已知生物标志物位置高度灵敏地检测这些甲基化变化。MSP需要多个样品制备步骤，包括蛋白质消化、DNA分离和检测前的亚硫酸氢盐转化。在这项工作中，我们提出了一个流线型的分析平台和仪器的集成所需的所有样品处理步骤，以获得定量MSP信号从原始生物样品通过使用液滴磁流体的原则。结合这个平台，我们提出了一个简化的协议，从细胞中的固相DNA提取和亚硫酸氢盐转化的基因组DNA，最大限度地减少了处理步骤和试剂体积的紧凑型检测平台上实施。
Early cancer detection requires identification of cellular changes resulting from oncogenesis. Abnormal DNA methylation patterns occurring early in tumor development have been widely identified as early biomarkers for multiple types of cancer tumors. Methylation-Specific PCR (MSP) has permitted highly sensitive detection of these methylation changes at known biomarker locations. MSP requires multiple sample preparation steps including protein digestion, DNA isolation, and bisulfite conversion prior to detection. In this work, we present a streamlined assay platform and instrumentation for integration of all sample processing steps required to obtain quantitative MSP signal from raw biological samples through the use of droplet magnetofluidic principles. In conjunction with this platform, we present a streamlined protocol for solid-phase DNA extraction from cells and bisulfite conversion of genomic DNA, minimizing the processing steps and reagent volume for implementation on a compact assay platform.