经典PCR
CHAPTER 1
The Basics of PCR and RT-PCR.. . 2
The Basics of PCR.. . . . . . . . . . . . . . 2
The Basics of RT-PCR.. . . . . . . . . . . 2
CHAPTER 2
Increasing RT-PCR Sensitivity.. . . . 3
Isolating High-Quality RNA .. . . . . . 3
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Using RNase H
Reverse Transcriptases . . . . . . . . . . . . . . . . . . . . . 4
Higher RT Incubation Temperatures . . . . . . . . . . . . . . . . . . . . . . . . 5
Additives to Enhance RT .. . . . . . . . . 5
RNase H Treatment .. . . . . . . . . . . . . 5
Improving Detection of Small Amounts of RNA . . . . . . . . . . . . . . 6
One-Step vs. Two-Step RT-PCR .. . . 6
CHAPTER 3
Improving RT-PCR Specificity.. . . . 7
Priming cDNA Synthesis.. . . . . . . . . 7
Higher RT Incubation Temperatures . . . . . . . . . . . . . . . . . . . . . . . . 7
Minimizing Contaminating Genomic DNA. . . . . . . . . . . . . . . . . . . 7
CHAPTER 4
RT-PCR Applications.. . . . . . . . . . . . 8
5′ and 3′ RACE .. . . . . . . . . . . . . . . . 8
Quantifying mRNA Expression.. . . . 8
CHAPTER 5
Improving PCR Specificity.. . . . . . 10
Primer Design.. . . . . . . . . . . . . . . . . 10
Primer Annealing Temperature .. . . 10
Touchdown PCR.. . . . . . . . . . . . . . . 11
Primer Concentration.. . . . . . . . . . . 11
Primer Purity and Stability.. . . . . . . 11
Hot Start.. . . . . . . . . . . . . . . . . . . . . 12
Magnesium Concentration.. . . . . . . 13
Additives to Enhance PCR.. . . . . . . 13
Nested PCR .. . . . . . . . . . . . . . . . . . 13
CHAPTER 6
Increasing PCR Sensitivity.. . . . . . 14
Template Quality .. . . . . . . . . . . . . . 14
Template Concentration.. . . . . . . . . 14
Enzyme Choice .. . . . . . . . . . . . . . . 14
CHAPTER 7
Improving Fidelity.. . . . . . . . . . . . . . 15
Enzymes with Proofreading.. . . . . . 15
Enzyme Mixes .. . . . . . . . . . . . . . . . 15
Other Parameters .. . . . . . . . . . . . . . 15
CHAPTER 8
Other Things to Consider.. . . . . . . 16
Amplifying Long Targets.. . . . . . . . 16
Prevention of Carry-Over Contamination . . . . . . . . . . . . . . . . . . . 16
Purification of PCR Products.. . . . . 16
CHAPTER 9
PCR Applications.. . . . . . . . . . . . . . . 17
Multiplex PCR .. . . . . . . . . . . . . . . . 17
Genotyping with Dinucleotide Repeat Markers . . . . . . . . . . . . . . 17
Tools for Detecting Polymorphisms. . . . . . . . . . . . . . . . . . . . . . . . 17
CHAPTER 10
Troubleshooting Guide.. . . . . . . . . . 18
CHAPTER 11
References.. . . . . . . . . . . . . . . . . . . . . 20
CHAPTER 12
Related Products.. . . . . . . . . . . . . . . 22
APPENDIX A
Selected Primer Sequences.. . . . . . 28
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