cfx96快速操作指南定義單核苷酸多態(tài)性

HRMPoweredbyPrecisionMelt Bio-Rad定義：單核苷酸多態(tài)性(singlenucleotidepolymorphisms,SNPs)主要是指在 SNPs研究進展和方 SNPs檢測方靈敏度和準確度的要求（反應原理嚴緊(操作和分析的自動化程度高SNP利用HRM鑒別已知的SNP多態(tài)1C/TandLargeVerySmall2C/Aand34SNPclassesdefinedbyVenteretal和SNPs檢測方法的分一、區(qū)分SNPs位點的方基于雜交的方基于酶的方直 的方

二、檢測分析技凝膠分析技熒光檢測技質譜檢測技原理不同而將SNPs位點檢測出來。（差異越大，檢測的1）利用固定 特異核苷酸探針(Allele-specificoligonucleotide，ASO)。PNALNAs 動態(tài)的加熱過程——動態(tài)等位 特異雜交(dynamicallele-specifichybridization,DASH)（Peptidenucleic 2、鏈擠入（形成三鏈復合結構受鹽濃度影響?。ǖ望}濃度的體系LNA(lockednucleicDNA、RNA或LNA結合（構象(dynamicallele-specific分子信標（Molecular蝎狀探針（Scorpion基于酶的DNA聚合4外切酶FEN）RNase1）DNA聚合等 特異性多重等 特異性 等 特異性Taqman 基本步驟（液相設計PCR擴增含SNPs位點的一段對PCR產物進行純化（去除引物和延伸產物檢測（放射性同位素標記法、發(fā)光檢測法、凝膠為基礎的熒光檢測法、質譜分析法、變性高壓液(arrayedprimerextension)——固 (PyrosequencingDNAATPATPsulfurylase(luciferaseapyrase原理：DNA聚合酶在一種dNTP的存在下進行引物延伸反2)連接聯(lián)合鏈式反應(combinedchainreaction連接-(ligation-rollingcircleamplication高分辨溶解技術HighResolutionMelt精確溶解分析軟件PrecisionMelt HighResolution陽性樣本可 HRMVersusMeltNormalizemeltApplyanoptionaltemperaturePlotcurvesinadifferencegraphforeasyClusterscurvesintogroupsrepresentingdifferent SYBR?vs.PCR抑 SYBR?

No

Source:Barrettetal., EvenDye

Source:Barrettetal., TheevendyeredistributiongivesequalsignalintensitiesfromdifferentsizedDNAMelt real-timePCR擴增完成后,使用“飽 ”進行溶解曲線分溶解溫DNAishalfdoubleandhalfsingle-Dependsonnucleotidecontentand

Melt MendelianGeneticsAllele

Homozygote

HomozygoteAllele C C AG HRM的應篩篩選突SNPDNA甲基化分物種鑒 鑒ScreeningforlossofAllelicprevalenceinaHLAcompatibility

IdentificationofcandidatepredispositionHRM實驗流AssayDesignSequence

Primer3,BLAST,

Evaluatereal-timeandHRM IdentifytherightSearchforSNPsbasedongene,locationorFindvariationsites(avoidvariationsthatimpactmeltMethylationCpGsitesinprimersandwithintheFragmentNCBISNPBalanceddistributionofG/CandA/TAnnealingbetweenat55-Nointernalsecondarystructures(hair-PrimerSimilarTms,within2-PrimerbindingAvoidtargetswithsecondaryAvoidLongerampliconsyieldmorecomplexprofiles,withmultiple s,considermelt complexity(M-fold)AvoidareasofsecondarystructureandhighGCLocalsequencecontextcaninfluencemutationDeterminefoldingcharacteristicsatannealingtemperature(DINAMelt)HRMHRMIncludecontrolsforeachknownvariantinthetestMakefreshreactionmixesforeachnew teswithintwohoursofPerformmeltcurve yfollowing反應過程MgCl2andbufferIntercalatingdyetypeandReactionMeltrampHRM Theobservedfluorescencecurveshowninblackisthecompositeofallfourpossibleduplexes AC GSetupreactionsusingHRMcompatibleSsoFASTEvaGreenRunamp+highresolutionmeltprotocolonCFX96or98°Cfor2min98°Cfor2-5s55-60°Cfor2-10sec( Gotostep2,39moretimes98°Cfor1min70°Cfor1Meltcurve75°Cto95°C0.2°C10sechold teInPrecisionMelt ysissoftware,importthedatafile(.pcrd)tocreateameltfile(.melt)DNA甲基2-5%ofthecytosinesinthegenomeare5thEpigeneticinformation-maychangeoverNoeffectonbaseMethylationCDH1(Cadherin113bpiQSYBRGreenMethylatedgDNAdilutedwithunmethylatedHRM smaspeciesidentificationwithSsoFastEvaGreenSupermixandPrecisionMelt ysisSoftwareSamplesamplifieddirectlyfromtissueculturerpoBgeneamplified(400to600bp4knownand10unknownM.M.M.M.M.

M.M.M.M.M.HRMAssaysConsistencyinreactionsetupandreagentuseisnecessaryforcomparisonsofsamplesProtocolsvarydependingontheResults