医学遗传学英文教学课件：Genetic Diagnosis-1 Methods in Molecular Medicine

GeneticDiagnosis-1:MethodsinMolecularMedicineTrendsofMedicinein21thCentury“5Pmedicine”:Prediction、Prevention、Participation、Personalization、PrecisionTreatmentafterwardPredictive&PreventiveMolecularepidemiology:Sub-populationRarediseases

CommondiseasesGeneralPersonalizedIntimesoftransformation,

weareallstudents！●predictindividualsusceptibilitytodisease,basedongenetic,genomic,andotherfactors;●providemoreusefultoolsandindividualizedprogramsfordiseaseprevention,basedonknowledgeofone’ssusceptibility;●detecttheonsetofdiseaseearlierandbeforeitisclinicallyevident,basedonnewlydiscoveredbiologicalmarkersthatarisefromchangesatthemolecularlevel;●preemptdiseaseprogression,asaresultofearlydetection;●targetmedicinesandtheirdosemorepreciselyandsafelytoeachpatient,onthebasisofadeepunderstandingofdiseasemechanismandtherolethatgeneticandgenomicfactorsplayintheindividualresponsetodrugs.----GinsburgGS,WillardHF.EssentialsofGenomicandPersonalizedMedicine.Elsevier,2010

InfectiousDisease

NeoplasticDisease

GeneticDisease

IdentityTesting

HLATyping PharmacogeneticsAreasofApplicationofGeneticDiagnosisPrevalenceofthecommonmonogeneticdisorders

inChina?---Noaccuratedata!~1%

inlivingbirth

---CarterCO.JMGenet1977;14:316TheestimatedaffectedneonatesinChina:

~160000peryear

!China‘sbirth

defect（出生缺陷）rate

（卫计委公布，Sep.,2012）

Birth

defect:Acongenitalabnormality（先天性畸形）.

Notallaredetectedatbirthorevensoonafter.~5.6%

~16millionneonates（新生儿）/yr→~900000affected,~270000visibleatbirthBeijing‘sbirth

defect

rate,2013

（北京市卫计委,Sep.11,2014）~14.68‰

forhouseholdregisteredneonates~26.22‰

fornon-householdregisteredneonatesHumangenomeDNAExtraction

Venousblood(5ml)→WBC→lysingbuffer(SDS)andproteaseK→centrifugation,collectsupernatant→phenol→centrifugation,collectsupernant→RNaseAandT1→phenol→centrifugation,collectsupernatant→potassiumacetateandEtOH(absolute)→genomicDNAappears(likecottonfiber)MillionsofthetargetsequencescanbereadilyobtainedbyPCRiftheflankingsequencesofthetargetareknown.3steps:DenatureRenature(annealing)ExtensionABI9700PCRSystemPCRamplificationofDNACycle

dscopies

3 24 45 810 25620 262,14430 268,435,456

PCRisusedinmanydiagnostictestsandforensictestsPointmutationscanbedetectedbyincorporatingthemintotheprimersPCRproductcanbesequencedSizingofrepeatedregions-microsatelliteDetectinginfectiousdiseases(viralgenomes)PCRispowerfulinlotsofbiomedicalfields!MullisK

(1993NP)分子杂交Sanger(dideoxy)sequencingFrederickSanger(1918-2013)

:NobelPrizeWinner,2times:

Pr.

Sequencing/1958;

DNASequencing/1980Mosaicism（嵌合体）(A)Sequenceanalysisoftheprobandrevealedahemizygousinsertionmutation(LAMP2/c.808dupG;p.A270Gfx3).(B)SequencesofDNAfromtheproband’smotherwerevariableandweaklysuggestiveofac.808dupG“G”peak.(C)SequencesofDNAfromunrelatedhealthycontrolswerenormal.

NGS(nextgenerationsequencing

)ASOdHPLC：WAVE系统的组成InterfacePumpDegasserDetectorColumnOvenAutosamplerTemperatureRackdHPLC高分辨率熔解曲线分析技术（highresolutionmelting，HRM）MLPAMLPAUsingmultiplexligation-dependentprobeamplification(MLPA)toidentifyanexon13deletionintheBRCA1gene.(A)Resultsfromacontrolsample.Numberedpeaksrepresentproductsfromeachexon;peakslabeledcarecontrolprobes.(B)ThesameanalysisonDNAfromapatientwithbreastcancer.Incomparisonwiththecontrolsample,theexon13peakisonlyhalfthesize.qPCR。A：TaqMan技术。B：real-timePCR分析结果。随着PCR循环次数（X轴）的增加，PCR产物（Y轴，以荧光强度表示。Rn即校正后的报告基因染色强度）呈指数性增长。

TheexpressionlevelsofthousandsofgenescanbesimultaneouslyanalyzedusingDNAmicroarraysThelevelatwhichageneisexpressed,asindicatedbymRNAquantities,canvarywidely,rangingfromnoexpressiontohundredsofmRNAcopiespercell.Gene-expressionpatternsvaryfromcelltypetocelltype.Evenwithinthesamecell,gene-expressionlevelsmayvaryasthecellrespondstochangesinphysiologicalcircumstances.DNAmicroarrays(genechips)DNA芯片进行基因表达定量分析。用颜色不同的荧光素分别标记实验标本和对照cDNA，然后混合，与固定在玻璃芯片表面的微阵列DNA探针进行杂交。数码成像的绿色点代表对照标本cDNA与探针杂交的信号，红色点代表实验标本cDNA与探针杂交的信号，黄色点代表对照和实验标本cDNA同时与探针杂交的信号，黑色区域代表对照和实验标本的cDNA均未与探针杂交。

MassSpectrometry（质谱法）-basedDiagnosticsMALDI-TOFMS：matrix-assistedlaserdesorption/ionization-timeofflightmassspectrometrySELDI-TOFMS：surface-enhancedlaserdesorption/ionization-timeofflightmassspectrometryRosenblattKP,etal.AnnuRevMed,2004,55:97–112PetricoinEF,etal.TrendsMolMed,2004,10:59–64荧光条形码标记的单分子检测技术（nCounterAnalysisSystem）：一种高通量检测基因表达谱、miRNA