cfx96快速操作指南定義單核苷酸多態(tài)性

HRMPoweredbyPrecisionMelt Bio-Rad定義：?jiǎn)魏塑账岫鄳B(tài)性(singlenucleotidepolymorphisms,SNPs)主要是指在 SNPs研究進(jìn)展和方 SNPs檢測(cè)方靈敏度和準(zhǔn)確度的要求（反應(yīng)原理嚴(yán)緊(操作和分析的自動(dòng)化程度高SNP利用HRM鑒別已知的SNP多態(tài)1C/TandLargeVerySmall2C/Aand34SNPclassesdefinedbyVenteretal和SNPs檢測(cè)方法的分一、區(qū)分SNPs位點(diǎn)的方基于雜交的方基于酶的方直 的方

二、檢測(cè)分析技凝膠分析技熒光檢測(cè)技質(zhì)譜檢測(cè)技原理不同而將SNPs位點(diǎn)檢測(cè)出來(lái)。（差異越大，檢測(cè)的1）利用固定 特異核苷酸探針(Allele-specificoligonucleotide，ASO)。PNALNAs 動(dòng)態(tài)的加熱過(guò)程——?jiǎng)討B(tài)等位 特異雜交(dynamicallele-specifichybridization,DASH)（Peptidenucleic 2、鏈擠入（形成三鏈復(fù)合結(jié)構(gòu)受鹽濃度影響?。ǖ望}濃度的體系LNA(lockednucleicDNA、RNA或LNA結(jié)合（構(gòu)象(dynamicallele-specific分子信標(biāo)（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(shè)計(jì)PCR擴(kuò)增含SNPs位點(diǎn)的一段對(duì)PCR產(chǎn)物進(jìn)行純化（去除引物和延伸產(chǎn)物檢測(cè)（放射性同位素標(biāo)記法、發(fā)光檢測(cè)法、凝膠為基礎(chǔ)的熒光檢測(cè)法、質(zhì)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(jìn)行引物延伸反2)連接聯(lián)合鏈?zhǔn)椒磻?yīng)(combinedchainreaction連接-(ligation-rollingcircleamplication高分辨溶解技術(shù)HighResolutionMelt精確溶解分析軟件PrecisionMelt HighResolution陽(yáng)性樣本可 HRMVersusMeltNormalizemeltApplyanoptionaltemperaturePlotcurvesinadifferencegraphforeasyClusterscurvesintogroupsrepresentingdifferent SYBR?vs.PCR抑 SYBR?

No

Source:Barrettetal., EvenDye

Source:Barrettetal., TheevendyeredistributiongivesequalsignalintensitiesfromdifferentsizedDNAMelt real-timePCR擴(kuò)增完成后,使用“飽 ”進(jìn)行溶解曲線分溶解溫DNAishalfdoubleandhalfsingle-Dependsonnucleotidecontentand

Melt MendelianGeneticsAllele

Homozygote

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

IdentificationofcandidatepredispositionHRM實(shí)驗(yàn)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(yīng)過(guò)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