PCR技术及其应用

Frequently used PCR methods
Overlap-extension PCR
Frequently used PCR methods
Nested PCR
Frequently used PCR methods
Real-Time PCR (quantitative PCR)
Detect fluorescence for each cycle
Iso-Thermal PCR
Iso-Thermal
Fast
Sensitive
LAMP / RPA
Loop-mediated isothermal amplification (LAMP)
Recombinase polymerase amplification
Two primers
Quantitativable
Quantitaive by amplification curve
Quanlity control
Real-Time PCR
double-stranded DNA-binding dye
Real-Time PCR (quantitative PCR)
Detect fluorescence for each cycle
PCR in forensic science
variable number of tandem repeats
Short Tandem Repeat Mitochondria Sequencing
PCR in forensic science
variable number of tandem repeats
Genetic testing
Tissue typing
Tumor target typing
Cell Free DNA analysis
major current techniques for analyzing fetal chromosomes
PCR in diagnosis of fetal heredopathia
Short Tandem Repeat
Mitochondria Sequencing
PCR in forensic science
Sex discernment
Y-chromosome specific DNA
Medical application of PCR
Medical applications
Fetal sex discernment
Procedure
DYS14 gene
SRY gene
DAZ gene
Initialization step
Denaturation step
Annealing step Elongation step Final elongation
20~40 Cycles
Exponential amplification
Leveling off stage Plateau
Final hold
Evolution of machines for PCR
3
1
2
4
PCR Optimization
Proofreading DNA Polymerase Pfu, KOD …
Contamination Polymerase errors
Hairpins
GC-rich Template
DMSO, betaine Redesign Primers
Primer Design: avoid failure of amplification
High difference between product and primer melting temperatures.
3'-end dimer between the Forward and Reverse primers.
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 Reagents
Template DNA Primers
dNTP DNA Polymerase
Buffer ealling
v.s.
DNA polymerase
Why primers are essential for DNA polymerase?
PCR procedure & stages
Reverse Transcription PCR
Real-Time PCR (quantitative PCR)
Overlap-extension PCR
Nested PCR
Isothermal PCR
Frequently used PCR methods
Reverse Transcription PCR
3'-end Forward Primer dimer.
Terminal stability of the Forward Primer is too high.
Primer Design: avoid failure of amplification
PCR methods
Frequently used PCR methods
Hot-Start Polymerase
Non-specific priming
Primer dimers Magnesium concentration Deoxynucleotides
PCR Optimization: Primer Design Softwares
Primer3: Command line style design tool
Baby gender blood tests
Fetal sex discernment
Chorionic villus sampling after the 11th~15th week of pregnancy risk of damage to the fetus Obstetric ultrasonography after the 13th week of pregnancy Accuracy: ~90% Cell-free fetal DNA after the 7th week of pregnancy Accuracy: ~98%
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
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
polymerase chain reaction (PCR)
Developed in 1983 by Kary Mullis
1993年的诺贝尔化学奖
Animation of PCR
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
Sensitive
Long Primers
Application of PCR
Application of PCR
Selective DNA isolation
Amplification and Quantification
PCR in forensic science
PCR in diagnosis of diseases