InventedbyK.Mullisinthe1980s

(NobelPrize)PolymeraseChainReaction,PCR聚合酶連鎖反應,PCR12/30/2024PCRallowsyoutoamplifyDNACanamplifyspecificgeneCanamplifyuptoabillion-fold!PartofthenucleotidesequencemustbeknownPCR:Purpose12/30/2024DNAtemplate(doublestranded)primers(singlestrandedDNA)DNApolymerasenucleotides(A,C,G,T)thermocyclerPCR:Requirements12/30/2024Thermocycler-controlledtemperature

-controlledrateoftemperaturechangePCR:Requirements12/30/2024Step1:SeparatethetwostrandsofDNAtemplateusuallyaround95CStep2:Cooltolettheprimersbindtothetemplatestrandsusuallyaround40to60CStep3:DNApolymeraseextendstheprimerstwonewcomplementaryDNAstrandssynthesized

usuallyaround72CPCR:3steps12/30/2024Taqpolymerase

Needheat-stableDNApolymerase:

Taqpolymerase

fromathermophilic(heat-loving)bacterianotinactivatedbyrepeatedheattreatmentsofPCRstep112/30/2024CyclesofPCR:12/30/2024◎Avoidcontamination◎Component dH2O 10XPCRbuffer dNTP senseprimer antisenseprimer TaqDNApolymerasePolymeraseChainReaction,PCR12/30/2024◎Negativecontrol/Positivecontrol/Exp.◎DetectPCRproducts EtBrstainedagarosegelelectrophoresis Southernblot◎Optimization enzyme dNTP Mg2+ Temp.ofannealing,extensionanddenaturation cycleno. primersPolymeraseChainReaction,PCR12/30/202412/30/202412/30/202412/30/2024Ifeachcycletakesabout5minutes,howmanycopiescanyoumakeinanhour?Answer:212PCR:Amplification12/30/2024MedicaldiagnosticsviralRNAorDNADNAfingerprintingForensics(legalscience)crimespaternitytestingEvolutionarystudiesHumanGenomeProjectPCR:Applications12/30/2024PCR:ApplicationsInfectiousdiseasesHepatitisvirus(HBV,HCV)Humanimmunodeficiencyvirus(HIV)12/30/2024PCR:Viraldetection12/30/2024DNA指紋親子/刑事鑑定12/30/2024前言12/30/2024衛星DNA重複序列DNA微衛星體迷你衛星體巨衛星體12/30/2024圖１１－１12/30/2024重複序列DNA二種主要重複序列DNA已被鑑定：１縱排重複序列的衛星DNA約佔人類基因體的１０％２散佈性重複序列DNA約佔人類基因體的５～２０％衛星DNA只存於真核生物，功能是在減數分裂時，染色體可經由這些同源互補的序列，配對在一起以及造成DNA重組的位置。變數縱排重複序列依長度分為：微衛星體(microsatellite)、迷你衛星體(minisatellites)、巨衛星體(macrosatellite)。12/30/2024表１１－１12/30/2024微衛星體12/30/2024迷你衛星體12/30/2024巨衛星體這些DNA序列是非常大的，長度約百萬氮鹼基，其長度使這些DNAs易被破壞或斷裂，因為大部分法醫學檢體之DNA都有斷裂的現象，因些巨衛星體不能在法醫工作中使用。12/30/2024圖１１－２12/30/2024圖１１－３12/30/2024圖１１－４12/30/202412/30/2024圖１１－５12/30/2024圖１１－６12/30/2024表１１－１12/30/2024限制片段長度多形性若對偶基因為異時，稱該基因為異型合子(heterozygous)。有時變異會影響限制酶切割的部位，即原有的切割序列，因為變異而消失，或新的限制酶切割位置可能產生。因此基於序列上的不同而導致有不同的切割點，造成不同大小的限制酶切割片斷，即稱限制片段長度多形性RFLPs(restrictionfragmentlengthpolymorphisms)。12/30/2024限制酶片段長度多形性RFLPMethodofRFLPAnalysis1.DNAExtraction2.DigestionofDNAwitharestrictionendonuclease12/30/2024RFLP(續)3.AgaroseGelElectrophoresis4.PreparationofaSouthernBlot12/30/20245.HybridizationwithaSingle-LocusProbe6.DetectionofRFLPsviaautoradiographyRFLP(續)12/30/20247.Re-probesouthernblotwithadditionalprobesThesetofAutoradssoobtainedisknownasa"DNAProfile."RFLP(續)12/30/2024圖１１－７12/30/2024圖１１－８12/30/2024圖１１－９12/30/2024聚合酶連鎖反應PCR的極端靈敏度使交叉污染成為一個非常嚴重的問題。極微小量的污染DNA，可能來自組織、液體、細菌和實驗室設備。在某些條件下，法院收集樣品時易造成交叉污染。污染的附加條帶將會是可見且可能混淆、毀壞結果的。因此小心選擇適當的操作條件及步驟、使用污染防治控制，將使報告書中的交叉污染被識別出來。12/30/2024圖１１－１０12/30/202412/30/2024粒線體DNA及Y染色體12/30/2024PCR-RFLP12/30/2024PCR-RFLP之應用12/30/2024親子鑑定RFLP12/30/2024ChildMolestationEvidenceInthiscasethedefendantwaschargedwithassaultinghisgirlfriend'syoungson.Theevidencewasasemenstainonthechild'sperson.Comparisonsweremadewith5differentRFLPprobes.Twoareshownbelow.Thedefendant'sreferenceprofilematchedtheevidenceforeachprobe.Thedefendantcouldnotbeexcludedasasourceoftheevidence,andeventuallyagreedtoapleabargain.12/30/2024Inthisstudy,10victimsofsexualassaultwhobecamepregnantshortlythereaftertookpartinacontrolledstudy(Hammondetal,JAMA273,1774,(1995).Priortotesting,theywereinterviewedaboutcontinuingthepregnancy.Allwomenhadaconsensualpartnerwhowasalsoapotentialfatheroftheunbornchild.