PCR与亲子刑事鉴定.ppt

Invented by K. Mullis in the 1980s (Nobel Prize)Polymerase Chain Reaction, PCR聚合酶連鎖反應, PCR10/2/2023 PCR allows you to amplify DNACan amplify specific geneCan amplify up to a billion-fold!Part of the nucleotide sequence must be knownPCR: Purpose10/2/2023 DNA template (double stranded)primers (single stranded DNA)DNA polymerasenucleotides (A,C,G,T)thermocyclerPCR: Requirements10/2/2023 Thermocycler- controlled temperature- controlled rate of temperature changePCR: Requirements10/2/2023 Step 1: Separate the two strands of DNA templateusually around 95 CStep 2: Cool to let the primers bind to the template strandsusually around 40 to 60 CStep 3: DNA polymerase extends the primers two new complementary DNA strands synthesized usually around 72 CPCR: 3 steps10/2/2023 Taq polymerase Need heat-stable DNA polymerase:  Taq polymerase from a thermophilic (heat-loving) bacterianot inactivated by repeated heat treatments of PCR step 110/2/2023 Cycles of PCR:10/2/2023 ◎ Avoid contamination◎ Component dH2O 10 X PCR buffer dNTP sense primer antisense primer Taq DNA polymerase Polymerase Chain Reaction, PCR10/2/2023 ◎ 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. primersPolymerase Chain Reaction, PCR10/2/2023 10/2/2023 10/2/2023 10/2/2023 If each cycle takes about 5 minutes, how many copies can you make in an hour?Answer: 212PCR: Amplification10/2/2023 Medical diagnosticsviral RNA or DNADNA fingerprintingForensics (legal science)crimespaternity testingEvolutionary studiesHuman Genome ProjectPCR: Applications10/2/2023 PCR: ApplicationsInfectious diseasesHepatitis virus (HBV, HCV)Human immunodeficiency virus (HIV)10/2/2023 PCR: Viral detection10/2/2023 DNA指紋親子/刑事鑑定10/2/2023 前言自１９６０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