《PCR技术及其应用》.ppt

Mitochondria Sequencing PCR in forensic science Short Tandem Repeat variable number of tandem repeats PCR in forensic science Sex discernment Y-chromosome specific DNA Medical application of PCR Medical applications Genetic testing Tissue typing Tumor target typing Cell Free DNA analysis major current techniques for analyzing fetal chromosomes PCR in diagnosis of fetal heredopathia Baby gender blood tests Fetal sex discernment Chorionic villus sampling Obstetric ultrasonography Cell-free fetal DNA after the 13th week of pregnancy Accuracy: ~90% risk of damage to the fetus after the 11th~15th week of pregnancy after the 7th week of pregnancy Accuracy: ~98% Fetal sex discernment PCR技术及其应用 What is PCR How to PCR Why do PCR Developed in 1983 by Kary Mullis polymerase chain reaction (PCR) 1993年的诺贝尔化学奖 Animation of PCR PCR Reagents Template DNA Primers dNTP DNA Polymerase Buffer Mg2+ Questions Annealling DNA polymerase v.s. Why primers are essential for DNA polymerase? Initialization step Denaturation step Annealing step Elongation step Final elongation Final hold PCR procedure stages 20~40 Cycles Exponential amplification Leveling off stage Plateau Evolution of machines for PCR 1 2 3 4 PCR Optimization Contamination Hairpins Polymerase errors Magnesium concentration Non-specific priming Primer dimers Deoxynucleotides GC-rich Template Proofreading DNA Polymerase Pfu, KOD … DMSO, betaine Redesign Primers Hot-Start Polymerase PCR Optimization: Primer Design Softwares Primer premier: e-PCR (NCBI) Oligo 7: Primer3: Command line style design tool Multi-Oligo design for probes or primers Single paired primers design for PCR Avoid non-specific amplification by blast Primers for amplification of human b-actin genomic DNA region Primer Design: avoid failure of amplification 5’-primer: stem-loop structure with high melting temperature High difference between product and primer melting temperatures. 3-end Forward Primer dimer. Primer Design: avoid failure of amplification 3-