PCR技术及其应用PPT课件

1、.,1,PCR技术在医学中的应用,医学分子细胞生物学研究方法,.,2,What is PCR,How to PCR,Why do PCR,.,3,Developed in 1983 by Kary Mullis,polymerase chain reaction (PCR),1993年的诺贝尔化学奖,.,4,Animation of PCR,.,5,PCR Reagents,Template DNA,Primers,dNTP,DNA Polymerase,Buffer,Mg2+,.,6,Questions,Annealling,DNA polymerase,v.s.,Why primers a

2、re essential for DNA polymerase?,.,7,Initialization step,Denaturation step,Annealing step,Elongation step,Final elongation,Final hold,PCR procedure & stages,2040 Cycles,Exponential amplification,Leveling off stage,Plateau,.,8,Evolution of machines for PCR,1,2,3,4,.,9,PCR Optimization,Contamination,H

3、airpins,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,.,10,PCR Optimization: Primer Design Softwares,Primer premier:,e-PCR (NCBI),Oligo 7:,Primer

4、3:,Command line style design tool,Multi-Oligo design for probes or primers,Single paired primers design for PCR,Avoid non-specific amplification by blast,.,11,Primers for amplification of human b-actin genomic DNA region,.,12,Primer Design: avoid failure of amplification,5-primer: stem-loop structur

5、e with high melting temperature,.,13,High difference between product and primer melting temperatures.,3-end Forward Primer dimer.,Primer Design: avoid failure of amplification,3-end dimer between the Forward and Reverse primers.,Terminal stability of the Forward Primer is too high.,.,14,Primer Desig

6、n: avoid failure of amplification,.,15,PCR methods,.,16,Isothermal PCR,Reverse Transcription PCR,Frequently used PCR methods,Real-Time PCR (quantitative PCR),Overlap-extension PCR,Nested PCR,.,17,Frequently used PCR methods,Reverse Transcription PCR,.,18,Overlap-extension PCR,Frequently used PCR met

7、hods,.,19,Nested PCR,Frequently used PCR methods,.,20,Frequently used PCR methods,Real-Time PCR (quantitative PCR),Detect fluorescence for each cycle,Quantitaive by amplification curve,Quanlity control,.,21,Real-Time PCR,double-stranded DNA-binding dye,.,22,Real-Time PCR (quantitative PCR),Detect fl

8、uorescence for each cycle,.,23,Real-Time PCR (quantitative PCR),Quanlity control by melting curve,.,24,Fluorescent reporter probe,Real-Time PCR,.,25,Real-Time PCR,Pathogen Diagnosis,microRNA Diagnosis,heredopathia Diagnosis,.,26,Iso-Thermal PCR,Iso-Thermal,Fast,Sensitive,LAMP / RPA,.,27,Loop-mediate

9、d isothermal amplification (LAMP),.,28,Recombinase polymerase amplification,Two primers,Quantitativable,Sensitive,Long Primers,.,29,Application of PCR,.,30,Application of PCR,Selective DNA isolation,Amplification and Quantification,PCR in diagnosis of diseases,PCR in forensic science,.,31,PCR in for

10、ensic science,variable number of tandem repeats,Mitochondria Sequencing,Short Tandem Repeat,.,32,Mitochondria Sequencing,PCR in forensic science,Short Tandem Repeat,variable number of tandem repeats,.,33,PCR in forensic science,Sex discernment,Y-chromosome specific DNA,.,34,Medical application of PC

11、R,.,35,Medical applications,Genetic testing,Tissue typing,Tumor target typing,.,36,Cell Free DNA analysis,.,37,major current techniques for analyzing fetal chromosomes,.,38,PCR in diagnosis of fetal heredopathia,.,39,Baby gender blood tests,.,40,Fetal sex discernment,Chorionic villus sampling,Obstetric ultrasonography,Cell-free fetal DNA,after the 13th week of pregnancy Accuracy: 90%,risk