分子诊断原理与技术(一)

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1、DNAmethylationConceptDetectionApplicationDNAmethylationinvolvestheadditionofamethylgrouptothe5positionofcytosine,whichoccursinthecontextofCpG(cytosinefollowedbyguanine)dinucleotides.Thismodificationcanbeinheritedthroughcelldivision.CpGsitesareregionsofDNAwhereacytosinenucleotideoccursnext

2、toaguaninenucleotideinthelinearsequenceofbasesalongitslength.CpGisshorthandfor"—C—phosphate—G—",thatis,cytosineandguanineseparatedbyaphosphate,whichlinksthetwonucleosidestogetherinDNA.CpGnotationisusedtodistinguishthislinearsequencefromthebase-pairingofcytosineandguanine.ThefrequencyofCpG

3、dinucleotidesinhumangenomesis1%.ThereareregionsoftheDNAthathaveahigherconcentrationofCpGsites,knownasCpGislands.ManygenesinmammaliangenomeshaveCpGislandsassociatedwiththestartofthegene.Becauseofthis,thepresenceofaCpGislandisusedtohelpinthepredictionandannotationofgenes.CpGIslands(CGI)sear

4、chOnlineResourcehttp://cpgislands.usc.edu/http://www.ebi.ac.uk/Tools/emboss/cpgplot/index.htmlMethods1Non-methylation-specificPCRbasedmethodsDirectsequencingPyrosequencingMethylation-sensitivesingle-strandconformationanalysis(MS-SSCA)Highresolutionmeltinganalysis(HRM)Methylation-sensitive

5、singlenucleotideprimerextension(MS-SnuPE)Base-specificcleavage/MALDI-TOF2Methylation-specificPCR(MSP)3Microarray-basedmethodsTreatmentofDNAwithbisulfite*convertscytosineresiduestouracil,butleaves5-methylcytosineresiduesunaffected.Thus,bisulfitetreatmentintroducesspecificchangesintheDNAseq

6、uencethatdependonthemethylationstatusofindividualcytosineresidues,yieldingsingle-nucleotideresolutioninformationaboutthemethylationstatusofasegmentofDNA.*亚硫酸氢盐DirectsequencingThefirstreportedmethodofmethylationanalysisusingbisulfite-treatedDNAutilizedPCRandstandarddideoxynucleotideDNAsequ

7、encingtodirectlydeterminethenucleotidesresistanttobisulfiteconversion.Primersaredesignedtobestrand-specificaswellasbisulfite-specific(i.e.,primerscontainingnon-CpGcytosinessuchthattheyarenotcomplementarytonon-bisulfite-treatedDNA),flanking(butnotinvolving)themethyla