PCR与亲子刑事鉴定

2022/12/7PCR與親子刑事鑑定Invented by K.Mullis in the 1980s(Nobel Prize)Polymerase Chain Reaction,PCR聚合酶連鎖反應聚合酶連鎖反應,PCR2022/12/7PCR與親子刑事鑑定 PCR allows you to amplify DNA Can amplify specific gene Can amplify up to a billion-fold!Part of the nucleotide sequence must be knownPCR:Purpose2022/12/7PCR與親子刑事鑑定 DNA template(double stranded)primers(single stranded DNA)DNA polymerase nucleotides(A,C,G,T)thermocyclerPCR:Requirements2022/12/7PCR與親子刑事鑑定Thermocycler-controlled temperature-controlled rate of temperature changePCR:Requirements2022/12/7PCR與親子刑事鑑定 Step 1:Separate the two strands of DNA template usually around 95 C Step 2:Cool to let the primers bind to the template strands usually around 40 to 60 C Step 3:DNA polymerase extends the primers two new complementary DNA strands synthesized usually around 72 CPCR:3 steps2022/12/7PCR與親子刑事鑑定Taq polymerase Need heat-stable DNA polymerase:Taq polymerase from a thermophilic(heat-loving)bacteria not inactivated by repeated heat treatments of PCR step 12022/12/7PCR與親子刑事鑑定Cycles of PCR:2022/12/7PCR與親子刑事鑑定 Avoid contamination ComponentdH2O 10 X PCR buffer dNTP sense primerantisense primer Taq DNA polymerase Polymerase Chain Reaction,PCR2022/12/7PCR與親子刑事鑑定 Negative control/Positive control/Exp.Detect PCR productsEtBr stained agarose gel electrophoresisSouthern blot Optimization enzymedNTPMg2+Temp.of annealing,extension and denaturationcycle no.primersPolymerase Chain Reaction,PCR2022/12/7PCR與親子刑事鑑定2022/12/7PCR與親子刑事鑑定2022/12/7PCR與親子刑事鑑定2022/12/7PCR與親子刑事鑑定 If each cycle takes about 5 minutes,how many copies can you make in an hour?Answer:212PCR:Amplification2022/12/7PCR與親子刑事鑑定 Medical diagnostics viral RNA or DNA DNA fingerprinting Forensics(legal science)crimes paternity testing Evolutionary studies Human Genome ProjectPCR:Applications2022/12/7PCR與親子刑事鑑定PCR:Applications Infectious diseases Hepatitis virus(HBV,HCV)Human immunodeficiency virus(HIV)2022/12/7PCR與親子刑事鑑定PCR:Viral detection2022/12/7PCR與親子刑事鑑定DNA指紋指紋親子親子/刑事鑑定刑事鑑定2022/12/7PCR與親子刑事鑑定前言自年代後期，蛋白質的多型性已被用來偵測個體之間的遺傳差異。使用這些蛋白質標誌最主要的問題是這些標誌因其有限的變異性，故有使用上的極限。當這些蛋白質標誌被用在法醫學上的判斷時，主要是用以排除某位疑犯的可能性，而非確認其有犯罪。因為，確認二個檢體相異的機率遠高於確認二個檢體是來自同一個人的機率。2022/12/7PCR與親子刑事鑑定衛星DNAv重 複 序 列 D N Av微衛星體v迷你衛星體v巨衛星體2022/12/7PCR與親子刑事鑑定圖2022/12/7PCR與親子刑事鑑定重複序列DNA二種主要重複序列DNA已被鑑定：縱排重複序列的衛星DNA約佔人類基因體的散佈性重複序列DNA約佔人類基因體的衛星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)。2022/12/7PCR與親子刑事鑑定表2022/12/7PCR與親子刑事鑑定微衛星體微衛星體是簡單重複序列或短縱向重複片段(STR)，在個體間存在著極大重複次數的差異。FBI就是利用13組充分了解的短縱向重複片段來進行DNA指紋圖譜鑑定。現今短縱向重複片段檢測幾乎已包含在所有法醫學的檢測當中。2022/12/7PCR與親子刑事鑑定迷你衛星體迷你衛星基因座共計約數千個，每個基因座有一明顯的重複單位。這些序列被使用發現散佈在整個基因體的迷你衛星體長度多型性，用於產生DNA指紋。2022/12/7PCR與親子刑事鑑定巨衛星體這些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。2022/12/7PCR與親子刑事鑑定圖2022/12/7PCR與親子刑事鑑定圖2022/12/7PCR與親子刑事鑑定圖2022/12/7PCR與親子刑事鑑定單一基因座微衛星體之變數縱排重複序列現今大多用聚合酶連鎖反應(PCR)來分析變數縱排重複序列圖譜。為了要產生帶有一個多基因座探針的差異性圖譜，至少需四或五個單一基因座PCR引子被使用。2022/12/7PCR與親子刑事鑑定圖2022/12/7PCR與親子刑事鑑定圖2022/12/7PCR與親子刑事鑑定表2022/12/7PCR與親子刑事鑑定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(restriction fragment length polymorphisms)。2022/12/7PCR與親子刑事鑑定限制酶片段長度多形性限制酶片段長度多形性RFLPMethod of RFLP Analysis1.DNA Extraction2.Digestion of DNA with a restriction endonuclease2022/12/7PCR與親子刑事鑑定RFLP(續續)3.Agarose Gel Electrophoresis4.Preparation of a Southern Blot2022/12/7PCR與親子刑事鑑定5.Hybridization with a Single-Locus Probe6.Detection of RFLPs via autoradiographyRFLP(續續)2022/12/7PCR與親子刑事鑑定7.Re-probe southern blot with additional probesThe set of Autorads so obtained is known as a DNA Profile.RFLP(續續)2022/12/7PCR與親子刑事鑑定圖2022/12/7PCR與親子刑事鑑定圖2022/12/7PCR與親子刑事鑑定圖2022/12/7PCR與親子刑事鑑定聚合酶連鎖反應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。2022/12/7PCR與親子刑事鑑定圖2022/12/7PCR與親子刑事鑑定單一核苷酸多形性可應用於：在族群中基因型的變異是如何對疾病表現型造成差異、生態研究的應用、演化生物學、利用生物資訊學中資訊探索分析而發展的單一核苷酸多形性資料庫、藥物基因體學。2022/12/7PCR與親子刑事鑑定粒線體DNA及Y染色體由粒線體DNA的資料顯示，所有不同的粒線體DNA序列在171,500年前都歸屬於同一個原始女性，這個拼湊而成的被稱為粒線體的夏娃。代表男性的染色體記錄了父系遺傳的演化史，如同粒線體的夏娃般，染色體亞當也可以追溯到35,00090,000年前的某個原始人男性。2022/12/7PCR與親子刑事鑑定PCR-RFLP2022/12/7PCR與親子刑事鑑定PCR-RFLP之應用之應用2022/12/7PCR與親子刑事鑑定親子鑑定親子鑑定RFLP2022/12/7PCR與親子刑事鑑定Child Molestation Evidence1.In this case the defendant was charged with assaulting his girlfriends young son.2.The evidence was a semen stain on the childs person.3.Comparisons were made with 5 different RFLP probes.Two are shown below.4.The defendants reference profile matched the evidence for each probe.5.The defendant could not be excluded as a source of the evidence,and eventually agreed to a plea bargain.2022/12/7PCR與親子刑事鑑定In this study,10 victims of sexual assault who became pregnant shortly thereafter took part in a controlled study(Hammond et al,JAMA 273,1774,(1995).Prior to testing,they were interviewed about continuing the pregnancy.All women had a consensual partner who was also a potential father of the unborn child.Typical results:Either inclusion of consensual partner or exclusion:Prenatal Diagnosis of Rape Victims2022/12/7PCR與親子刑事鑑定Nested PCR2022/12/7PCR與親子刑事鑑定TA cloning2022/12/7PCR與親子刑事鑑定Reverse Transcription PCR2022/12/7PCR與親子刑事鑑定Real-time PCR