Bisulfite sequencing of DNA.
Bisulfite sequencing of DNA.
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DNA的亚硫酸氢盐测序。
DOI：
10.1002/0471142727.mb0709s91
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发表时间：
2010-07
影响因子：
--
通讯作者：
Kladde, Michael P
中科院分区：
文献类型：
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作者：
Darst, Russell P;Pardo, Carolina E;Ai, Lingbao;Brown, Kevin D;Kladde, Michael P
5-甲基胞嘧啶（m5 C）在单链DNA上的精确位置可以通过亚硫酸氢盐基因组测序（BGS）来确定。用亚硫酸氢根离子处理优先使未甲基化的胞嘧啶脱氨基，然后在磺化后转化为尿嘧啶。从脱氨基的DNA和从扩增子克隆的测序产物扩增感兴趣的区域允许以单核苷酸分辨率沿沿着单个DNA分子测定甲基化，这是用其他甲基化分析技术不可能的。本单元描述了一种适用于大多数DNA来源的BGS技术，包括甲醛固定的组织。考虑实验设计和常见的误差来源进行了讨论。
Exact positions of 5-methylcytosine (m5C) on a single strand of DNA can be determined by bisulfite genomic sequencing (BGS). Treatment with bisulfite ion preferentially deaminates unmethylated cytosines, which then convert to uracil upon desulfonation. Amplifying regions of interest from deaminated DNA and sequencing products cloned from amplicons permits determination of methylation at single nucleotide resolution along single DNA molecules, which is not possible with other methylation analysis techniques. This unit describes a BGS technique suitable for most DNA sources, including formaldehyde-fixed tissue. Considerations for experimental design and common sources of error are discussed.