Tost Jörg, Gut Ivo G
Laboratory for Epigenetics, CEA-Institut de Génomique, Centre National de Génotypage, Evry Cedex, France.
Nat Protoc. 2007;2(9):2265-75. doi: 10.1038/nprot.2007.314.
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.
焦磷酸测序是一种合成测序方法，它通过将释放的焦磷酸酶促转化为成比例的光信号来定量监测核苷酸的实时掺入。在各种发育和病理情况下，定量测量对于DNA甲基化分析尤为重要。通过焦磷酸测序分析DNA甲基化模式，将简单的反应方案与对几个紧密相邻的CpG位点甲基化程度的可重复且准确的测量相结合，具有高定量分辨率。经过亚硫酸氢盐处理和PCR后，根据T和C的比例确定序列中每个CpG位置的甲基化程度。对于同一模板，可以重复纯化和测序过程，以分析同一扩增产物中的其他CpG位点。当以亚硫酸氢盐处理的DNA作为起始材料时，使用该方案大约4小时内可为多达96个DNA样本获得定量表观基因型。
