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The first step to model DTG-PCR
Ji Youn Lee
Cell and microbial engineering laboratory
Seoul National University
Basic concept
• Make use of the math in published ones
: Competitive PCR
• Addition of the ssDNA distribution profile with
temperature
• Experimental part
• Modeling and simulation part
Competitive PCR
• Originally introduced for the analysis of
quantitative PCR
• Target and various concentrations of internal
standard
• Efficiencies at each cycle
eiT and eiS
Ti = (1+ eiT ) Ti-1
Si = (1+ eiS ) Si-1
Tn = P i=1 n (1+ eiT ) T0
Sn = P i=1 n (1+ eiS ) S0
Our modeling ?
• Factors affecting PCR
– the concentrations of DNA polymerase, dNTPs, MgCl2,
DNA and primers
– the denaturing annealing and synthesis temperatures
– the length and number of cycles
– ramping times and the presence of contaminating DNA
and inhibitors in the sample
• Modeling
– Exceptions
Enzyme inactivation
Substrate consumption
High-tolerance of PCR to mismatches
– All together~
Each steps
• 1st step: denaturation
dsDNA  ssDNA
Number of ssDNA molecules participating in the annealing
reaction (melting curve or data table)
• 2nd step: annealing
ssDNA + primer  heteroduplex
• 3rd step: extension
heteroduplex + polymerase  enzyme-substrate complex
[complex + dNTP  elongated complex]repeat
fully elongated complex  dsDNA + polymerase
 enzyme kinetics 이용 (Michealis-Menten equation)
Work to do..
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Experiments
Melting curve or melting data acquisition
Equations
Search the kinetic parameters