The first step to model DTG-PCR Ji Youn Lee Cell and microbial engineering laboratory Seoul National University.

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Presentation transcript:

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  i T and  i S T i = (1+  i T ) T i-1 T n =  i=1 n (1+  i T ) T 0 S i = (1+  i S ) S i-1 S n =  i=1 n (1+  i S ) S 0

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 1 st step: denaturation dsDNA  ssDNA Number of ssDNA molecules participating in the annealing reaction (melting curve or data table) 2 nd step: annealing ssDNA + primer  heteroduplex

3 rd 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.. Experiments Melting curve or melting data acquisition Equations Search the kinetic parameters