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1、2019/5/14,PCR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, PCR,2019/5/14,PCR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 known,PCR: Purpose,2019/5/14,PCR與親子
2、刑事鑑定,DNA template (double stranded) primers (single stranded DNA) DNA polymerase nucleotides (A,C,G,T) thermocycler,PCR: Requirements,2019/5/14,PCR與親子刑事鑑定,Thermocycler - controlled temperature - controlled rate of temperature change,PCR: Requirements,2019/5/14,PCR與親子刑事鑑定,Step 1: Separate the two str
3、ands 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 C,PCR: 3 steps,2019/5/14,PCR與親子刑事鑑定,Taq polymerase,Need heat-stab
4、le DNA polymerase: Taq polymerase from a thermophilic (heat-loving) bacteria not inactivated by repeated heat treatments of PCR step 1,2019/5/14,PCR與親子刑事鑑定,Cycles of PCR:,2019/5/14,PCR與親子刑事鑑定, Avoid contamination Component dH2O 10 X PCR buffer dNTP sense primer antisense primer Taq DNA polymerase,Po
5、lymerase Chain Reaction, PCR,2019/5/14,PCR與親子刑事鑑定, Negative control / Positive control / Exp. Detect PCR products EtBr stained agarose gel electrophoresis Southern blot Optimization enzyme dNTP Mg2+ Temp. of annealing, extension and denaturation cycle no. primers,Polymerase Chain Reaction, PCR,2019/
6、5/14,PCR與親子刑事鑑定,2019/5/14,PCR與親子刑事鑑定,2019/5/14,PCR與親子刑事鑑定,2019/5/14,PCR與親子刑事鑑定,If each cycle takes about 5 minutes, how many copies can you make in an hour? Answer: 212,PCR: Amplification,2019/5/14,PCR與親子刑事鑑定,Medical diagnostics viral RNA or DNA DNA fingerprinting Forensics (legal science) crimes pa
7、ternity testing Evolutionary studies Human Genome Project,PCR: Applications,2019/5/14,PCR與親子刑事鑑定,PCR: Applications,Infectious diseases Hepatitis virus (HBV, HCV) Human immunodeficiency virus (HIV),2019/5/14,PCR與親子刑事鑑定,PCR: Viral detection,2019/5/14,PCR與親子刑事鑑定,DNA指紋,親子/刑事鑑定,2019/5/14,PCR與親子刑事鑑定,前言,自年
8、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。,2019/5/14,PCR與親子刑事鑑定,衛星DNA,重複序列DNA 微衛星體 迷你衛星體 巨衛星體,2019/5/14,PCR與親子刑事鑑定,圖,2019/5/14,PCR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
9、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)。,2019/5/14,PCR與親子刑事鑑定,表,2019/5/14,PCR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
10、測當中。,2019/5/14,PCR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。,2019/5/14,PCR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。,2019/5/14,PCR與親子刑事鑑定,圖,2019/5/14,PCR與親子刑事鑑定,圖,2019/5/14,PCR與親子刑事鑑定,圖,2019/5/14,PCR與親子刑事鑑定
11、,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。,2019/5/14,PCR與親子刑事鑑定,圖,2019/5/14,PCR與親子刑事鑑定,圖,2019/5/14,PCR與親子刑事鑑定,表,2019/5/14,PCR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
12、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)。,2019/5/14,PCR與親子刑事鑑定,限制酶片段長度多形性RFLP,Method of RFLP Analysis,1.DNA Extraction,2.Digestion of DNA with a restriction endonuclease,2019/5/14,PCR與親子刑事鑑定,RFLP(續),3.Agarose Gel Electrophoresis,4.Preparation of a Southern Blot,2019/5
13、/14,PCR與親子刑事鑑定,5.Hybridization with a Single-Locus Probe,6.Detection of RFLPs via autoradiography,RFLP(續),2019/5/14,PCR與親子刑事鑑定,7.Re-probe southern blot with additional probes,The set of Autorads so obtained is known as a “DNA Profile.“,RFLP(續),2019/5/14,PCR與親子刑事鑑定,圖,2019/5/14,PCR與親子刑事鑑定,圖,2019/5/14,
14、PCR與親子刑事鑑定,圖,2019/5/14,PCR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。,2019/5/14,PCR與親子刑事鑑定,圖,2019/5/14,PCR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
15、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。,2019/5/14,PCR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。,2019/5/14,PCR與親子刑事鑑定,PCR-RFLP,2019/5/14,PCR與親子刑事鑑定,PCR-RFLP之應用,2019/5/14,PCR與親子刑事鑑定,親子鑑定 RFLP,2019/5/1
16、4,PCR與親子刑事鑑定,Child Molestation Evidence In this case the defendant was charged with assaulting his girlfriends young son. The evidence was a semen stain on the childs person. Comparisons were made with 5 different RFLP probes. Two are shown below. The defendants reference profile matched the evidenc
17、e for each probe. The defendant could not be excluded as a source of the evidence, and eventually agreed to a plea bargain.,2019/5/14,PCR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).
18、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 Victims,2019/5/14,PCR與親子刑事鑑定,Nested PCR,2019/5/14,PCR與親子刑事鑑定,TA cloning,2019/5/14,PCR與親子刑事鑑定,Reverse Transcription PCR,2019/5/14,PCR與親子刑事鑑定,Real-time PCR,