PCR技术及其应用 ppt课件

Iso-Thermal PCR
Iso-Thermal Fast Sensitive LAMP / RPA
Loop-mediated isothermal amplification (LAMP)
Recombinase polymerase amplification
Two primers Quantitativable Sensitive Long Primers
Primer premier: Multi-Oligo design for probes or primers
Oligo 7: Single paired primers design for PCR
e-PCR (NCBI) Avoid non-specific amplification by blast
Proofreading DNA Polymerase Pfu, KOD …
DMSO, betaine Redesign Primers
Hot-Start Polymerase
PCR Optimization: Primer Design Softwares
Primer3: Command line style design tool
PCR Reagents
Template DNA Primers
dNTP DNA Polymerase
Buffer Mg2+
Questions
Annealling
v.s.
DNA polymerase
Why primers are essential for DNA polymerase?
PCR procedure & stages
Real-Time PCR (quantitative PCR) Quanlity control by melting curve
Real-Time PCR Fluorescent reporter probe
Real-Time PCR
Pathogen Diagnosis microRNA Diagnosis heredopathia Diagnosis
PCR技术及其应用
医学分子细胞生物学研究方法
PCR技术在医学中的应用
What is PCR Why do PCR How to PCR
polymeR)
Developed in 1983 by Kary Mullis
1993年的诺贝尔化学奖
Animation of PCR
Primer Design: avoid failure of amplification High difference between product and primer melting temperatures.
3'-end dimer between the Forward and Reverse primers. 3'-end Forward Primer dimer. Terminal stability of the Forward Primer is too high.
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
3 Evolution of machines for PCR
1
2
4
PCR Optimization
Contamination Polymerase errors Hairpins GC-rich Template Non-specific priming Primer dimers Magnesium concentration Deoxynucleotides
Frequently used PCR methods Reverse Transcription PCR
Frequently used PCR methods Overlap-extension PCR
Frequently used PCR methods Nested PCR
Frequently used PCR methods Real-Time PCR (quantitative PCR)
Application of PCR
Application of PCR
Selective DNA isolation Amplification and Quantification PCR in forensic science PCR in diagnosis of diseases
Detect fluorescence for each cycle Quantitaive by amplification curve Quanlity control
Real-Time PCR double-stranded DNA-binding dye
Real-Time PCR (quantitative PCR) Detect fluorescence for each cycle
Initialization step Denaturation step
20~40 Cycles
Annealing step
Elongation step
Final elongation
Final hold
Exponential amplification Leveling off stage Plateau
Primer Design: avoid failure of amplification
PCR methods
Frequently used PCR methods
Reverse Transcription PCR Real-Time PCR (quantitative PCR) Overlap-extension PCR Nested PCR Isothermal PCR