甲基化檢測(cè)20121224課件

1、MLH1甲基化檢測(cè)MLH1甲基化測(cè)序摘要CpG島的概念、意義PCR的原理及基本流程MSP的原理、流程BSQ/Methylight的技術(shù)簡(jiǎn)介CpG dinucleotides and CpG-islandsCpG-islands(ever)the length of genomic region200bpG+C 50%observed/expected CpG 60%CpG-islands(most recently-to exclude Alu)the length of genomic region500bpG+C 55% observed/expected CpG 65% CpG dinu

2、cleotide-CG.CG- -GC.GC-Wild-typegenerally(70%)methylationgenerallyunmethylationJournal of Molecular Diagnostics(2009)Antonia R ,Sepulveda ,et al.CpG island methylationCpG島多位于基因啟動(dòng)子區(qū)域，當(dāng)其發(fā)生甲基化時(shí)將改變?nèi)旧|(zhì)結(jié)構(gòu)、抑制轉(zhuǎn)錄因子結(jié)合從而抑制基因的表達(dá)后天表觀遺傳改變CpG-islands MLH1 methylationbiallic methylated MLH1loss of expressiondefetio

3、n of MMRCRC inition and progressionunmethylated MLH1MSIThe Molecular Differnces between Sprodic MSI and LSSprodic MSILSMLH1 epimutationMLH1 MSH2 PMS2 MSH6 mutation40%50% B-raf mutationNone Arch Pathol Lab Med.2011Katherine B. Geiersbach et al.PCRPCR-即聚合酶鏈?zhǔn)椒磻?yīng)，是指在DNA聚合酶催化下，以母鏈DNA為模板，以特定引物為延伸起點(diǎn)，通過(guò)變性、退火

4、、延伸等步驟，體外復(fù)制出與母鏈摸板互補(bǔ)的子鏈DNA過(guò)程。反應(yīng)成分：模板DNA、引物、dNTP、DNA聚合酶、緩沖液、Mg2+等。PCR反應(yīng)基本步驟：1、變性：高溫下，雙鏈DNA解離成單鏈；2、退火：低溫下，引物與模版DNA互補(bǔ)區(qū)結(jié)合3、延伸：中溫下，DNA聚合酶催化以引物為起始點(diǎn)的 DNA鏈延伸反應(yīng)。MSP 流程DNA 提取亞硫酸鹽修飾*PCR*瓊脂糖凝膠電泳亞硫酸鹽修飾PCR引物設(shè)計(jì)PCRMLH1引物比較The Herman primer set not specific for a more proximal region of the promoter (A region)may wro

5、ngly estimated the association between methylation of MLH1 and the lack of its protein expressionThe Paker/MJ primer sets specific for a more proximal region of the promoter (C region)more closely with loss of MLH1 expressionOncogene (2007) 26, 75967600E Capel, R Hamelin et al.PCR conditionHerman95

6、for 15 min;35 cycles of 95 for 30 s,59 for 45 s and 72 for 30 s ; the final extension 72for 4 min .Parker95 for 15 min; 40 cycles of 95 for 30 s,53 for 45 s and 72 for 30 s;the final extension 72 for 4 min.MJ95 for 15 min; 40 cycles of 95 for 30 s,57 for 45 s and 72 for 30 s;the final extension 72 f

7、or 4 min.Oncogene (2007) 26, 75967600E Capel, R Hamelin et al.With HotstarTaq DNA polymerase (Cat. No. 203203)根據(jù)不同聚合酶的特點(diǎn)以及實(shí)驗(yàn)實(shí)際結(jié)果可進(jìn)行溫度、時(shí)間調(diào)節(jié)瓊脂糖凝膠電泳(2%)結(jié)果解讀(Herman)115bp124bpBisulfite Sequencing技術(shù)流程DNA提取亞硫酸鹽修飾PCR*測(cè)序*(克隆/直接)數(shù)據(jù)分析只設(shè)計(jì)一對(duì)引物，不包含CpG位點(diǎn)與MSP比較甲基化檢測(cè)方法比較MSPBSQMethylight優(yōu)點(diǎn)敏感、快速、經(jīng)濟(jì)、實(shí)用、應(yīng)用范圍廣可靠、定量精確(單堿基)高通量、高敏感、所需模板量少、污染少、操作誤差小缺點(diǎn)引物設(shè)計(jì)復(fù)雜、僅定性分析、需避