焦测序
照明菌甲基化试验
亚硫酸氢盐测序
DNA甲基化
亚硫酸氢盐
甲基化
生物
DNA测序
CpG站点
计算生物学
表观遗传学
DNA
分子生物学
差异甲基化区
RNA导向的DNA甲基化
遗传学
化学
甲基化DNA免疫沉淀
表观遗传学
基因
基因表达
出处
期刊:Nature Protocols
[Springer Nature]
日期:2007-09-01
卷期号:2 (9): 2265-2275
被引量:580
标识
DOI:10.1038/nprot.2007.314
摘要
Pyrosequencing is a sequencing-by-synthesis method that quantitatively monitors the real-time incorporation of nucleotides through the enzymatic conversion of released pyrophosphate into a proportional light signal. Quantitative measures are of special importance for DNA methylation analysis in various developmental and pathological situations. Analysis of DNA methylation patterns by pyrosequencing combines a simple reaction protocol with reproducible and accurate measures of the degree of methylation at several CpGs in close proximity with high quantitative resolution. After bisulfite treatment and PCR, the degree of each methylation at each CpG position in a sequence is determined from the ratio of T and C. The process of purification and sequencing can be repeated for the same template to analyze other CpGs in the same amplification product. Quantitative epigenotypes are obtained using this protocol in approximately 4 h for up to 96 DNA samples when bisulfite-treated DNA is already available as the starting material.
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