PCR技术及其应用ppt课件.ppt

PCR技术在医学中的应用
医学分子细胞生物学研究方法
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What is PCR
How to PCR
Why do PCR
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Developed in 1983 by Kary Mullis
polymerase chain reaction (PCR)
1993年的诺贝尔化学奖
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Animation of PCR
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PCR Reagents
Template DNA
Primers
dNTP
DNA Polymerase
Buffer
Mg2+
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Questions
Annealling
DNA polymerase
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Why primers are essential for DNA polymerase?
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Initialization step
Denaturation step
Annealing step
Elongation step
Final elongation
Final hold
PCR procedure & stages
20~40 Cycles
Exponential amplification
Leveling off stage
Plateau
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Evolution of machines for PCR
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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
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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
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