Microdroplet PCR for Highly Multiplexed Targeted Bisulfite Sequencing.
Microdroplet PCR for Highly Multiplexed Targeted Bisulfite Sequencing.
复制标题
用于高度多重靶向亚硫酸氢盐测序的微滴 PCR。
DOI：
10.1007/978-1-4939-7481-8_17
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发表时间：
2018
期刊：
影响因子：
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通讯作者：
Salomon,DanielR
中科院分区：
文献类型：
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作者：
Komori,HKiyomi;LaMere,SarahA;Hart,Traver;Head,StevenR;Torkamani,Ali;Salomon,DanielR
存在许多检查 CpG DNA 甲基化的方法。然而，其中许多都是定性的，很难同时应用于大量基因，或者不容易针对特定的感兴趣区域。基于微滴 PCR 的亚硫酸氢盐测序可对研究者选定的感兴趣区域进行定量单碱基分辨率分析。基因组 DNA 进行亚硫酸氢盐转化后，使用定制引物库进行靶向微滴 PCR。然后将样品片段化、串联并通过高通量测序进行测序。最新的技术允许使用少至 250 ng 的亚硫酸氢盐转化的 DNA 来进行该方法。该方法的主要优点是能够手动选择由多达 10,000 个 500-600 bp 的扩增子覆盖的目标区域。此外，微滴 PCR 的性质实际上消除了 PCR 偏差，并允许在单个管中同时扩增所有目标。
Many methods exist for examining CpG DNA methylation. However, many of these are qualitative, laborious to apply to a large number of genes simultaneously, or are not easy to target to specific regions of interest. Microdroplet PCR-based bisulfite sequencing allows for quantitative single base resolution analysis of investigator selected regions of interest. Following bisulfite conversion of genomic DNA, targeted microdroplet PCR is conducted with custom primer libraries. Samples are then fragmented, concatenated, and sequenced by high-throughput sequencing. The most recent technology allows for this method to be conducted with as little as 250 ng of bisulfite-converted DNA. The primary advantage of this method is the ability to hand-select the targeted regions covered by up to 10,000 amplicons of 500–600 bp. Moreover, the nature of microdroplet PCR virtually eliminates PCR bias and allows for the amplification of all targets simultaneously in a single tube.