【正文】 ve only! ? Cloned genomic sequencing (Sanger) – quantitative analysis of multiple adjacent CpG sites – Highly labor intensive! ? Methylationspecific PCR – rapid method for initial screen of samples – Only qualitative! ? These account for ~90% of all studies today Oligonucleotides as a model system – measured Pyrograms 0% mC oligo in PCR 50% mC oligo in PCR 100% mC oligo in PCR 0% 0% 0% 0% AC/TGTC/TGACGC/TGTATTC/TGGG Pos1 Pos2 Pos3 Pos4 % % 100% % % % % % 1. Why Pyrosequencing? (cont’d) Confidently measure the individual degree of methylation in adjacent CpG sites in one easy analysis Pos 1 Pos 2 Pos 3 Pos 4 Pos 5 % methylation at individual CpG sites Pos 2 Pos 1 Pos 3 Pos 4 Pos 5 Mean value 6 replicates CV % 60,12 56,85 66,85 55,87 63,15 2,9 2,6 2,7 1,7 2,9 2. Why Pyrosequencing? Builtin QC through sequence context Sequence: C/TGGGGC/TGAGATTATC/TGTC/TGATAGC/TGGGGG 3. Why Pyrosequencing? Builtin QC for pletion of the bisulfite treatment Before bisulfite treatment: After bisulfite treatment and PCR: GCGGTCAGTGACGCGATGGAGCGGGC GYGGTTAGTGAYGYGATGGAGYGGGT Any C not followed by a G gives bisulfite QC 4. Why Pyrosequencing? Flexibility in sequencing primer position opens the possibility to analyse any CpG sites you like agggagttgggatttttgtattgYggtaaataagta YgtttgYgYggtYgtagaggtaggttggYgYgtatgtttaggYggggatgtgtgYgaagtttgtYgttgttgtagYgagtttggYgtagagtggagYggtYgtYggagatgtttgaYgtatttgtttgaggagYggttagtgaYgYgatggagYgggtaaggttagttgtgtYggtggttttttttaagagatagtttgggg Sequencing primers Five different sequencing primers tested for this amplicon. All five worked perfectly. ?一個反應(yīng)檢測多個甲基化位點 ?測定甲基化的程度 ?無須重復(fù)試驗 ?結(jié)果準(zhǔn)確 Pyrosequencing在基因甲基化研究中的優(yōu)勢 英國醫(yī)學(xué)刊物 《 手術(shù)小刀 Lancet》 新型大便測試法出世 醫(yī)生可以檢測大便診斷癌癥 結(jié)腸癌是工業(yè)化世界最為常見的一種癌癥，目前一般只能在癌變部位惡化到病人有明顯不適的感覺時才能被發(fā)現(xiàn)、確癥。 長期以來，科學(xué)家們都試圖在癌癥患者的大便中找出相應(yīng)的表現(xiàn)病變的征兆 (物質(zhì) )，以減少入侵性的體內(nèi)測試 奧地利因斯布魯克醫(yī)學(xué)院的專家們早就可以通過檢測大便中發(fā)現(xiàn)的 DNA的化學(xué)變化，把健康人的大便與結(jié)腸癌患者的大便區(qū)分開。 有一種叫做 SFRP2的基因，在癌癥患者的大便中更容易被 甲基化 。研究者說，大便標(biāo)本中 SFRP2 甲基化的程度是癥斷結(jié)腸癌的最靈敏的標(biāo)記。 專家們還將進(jìn)一步進(jìn)行研究，看能不能在大便中找出一組標(biāo)記，以便在更早的時候確癥。 Currently available Pyrosequencing assays ? Developmental disorders PraderWilli / Angelman ? Tumorigenesis MLH1 ? X chromosome inactivation ARAF1 XIST ? More in the pipeline Colella S., Shen L., Baggerly ., Issa ., Krahe R. Sensitive and quantitative universal Pyrosequencing methylation analysis of CpG sites. BioTechniques 35 146150 (July 2022). Tost J., Dunker J., Glynne Gut Y. Analysis and quantification of multiple methylation variable positions in CpG islands by Pyrosequencing. BioTechniques 35:152156 (July 2022).* Goossens D., Van Gestel S., Claes S., De Rijk P., D Souery D., Massat I., Van den Bossche D., Backhovens H., Mendlewicz J., Van Broeckhoven C., DelFavero J. A novel CpGassociated brainexpressed candidate gene for chromosome 18qlinked bipolar disorder. Molecular Psychiatry (2022) 8, 8389. Uhlmann K., Brinckmann A., Toliat M. R., Ritter H., N252。rnberg P. Evaluation of a potential epigeic biomarker by quantitative methylsingle nucleotide polymorphism analysis. Electrophoresis 2022, 23, 40724079. 利用 Pyrosequencing研究甲基化的研究論文 01020304050607080901002022 2022 2022 2022利用 Pyrosequencing發(fā)表的國際論文數(shù)量 () 0246810122022 2022 2022 2022發(fā)表文章數(shù)目逐年快速遞增 發(fā)表在 Nature/Science文章數(shù)目逐年快速遞增 一個應(yīng)用越來越廣泛的遺傳學(xué)研究新工具 300多篇文獻(xiàn)詳見 : ?Pyrosequencing技術(shù)原理及特色應(yīng)用 2/ ?Pyrosequencing技術(shù)在分子診斷方面的應(yīng)用舉例 / ?如欲了解詳細(xì)信息和最新應(yīng)用，歡迎訪問： Pyrosequencing的部分用戶 CuraGen Corporation Lynx Therapeutics Howard University Genzyme Genomics Collaborative NIH UC Davis Harvard Center for Cancer Prevention Yale University Hospital for Sick Children Max Planck Institute Baylor College of Medicine RIKEN AstraZeneca GlaxoSmithKline Merck ScheringPlough Abbott Janssen Pharmaceutica, NV Bayer AG Biogen Karolinska Institute NCI DuPont Agriculture German Armed Forces NASA 第四軍醫(yī)大學(xué)全軍基因診斷中心 中科院健康中心 上海晶泰生物技術(shù)有限公司 全球用戶 300 customers 25 accounts with 2 or more systems Let Us Show You the Light. The End Thank You!