PCR 의 kinetics 에 대한 좀더 많은 생각 … 2003 년 12 월 16 일 이지연.

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Presentation transcript:

PCR 의 kinetics 에 대한 좀더 많은 생각 … 2003 년 12 월 16 일 이지연

PCR Plateau Phase A number of factors –Utilization of substrates (dNTPs or primers) –Thermal inactivation and limiting concentration of DNA polymerase –Inhibition of enzyme activity by increasing pyrophosphate concentration –Reannealing of specific product at concentrations above M –Reduction in the denaturation efficiency per cycle –Destruction of product due to Taq DNA polymerase 5’  3’ exonulease activity

Related Work I Biochemica et Biophysica Acta 1494 (2000) Simulation of later cycle conditions –1kb dsDNA addition –A 30-fold molar excess of fragments to polymerase molecules almost completely inhibited the amplification of target stands. –The main factor contributing to the plateau phase in PCR consists of binding of DNA polymerase to its amplification products. –Taq and Tfl DNA polymerases bind to short double-stranded DNA fragments without sequence specificity

Related Work II Biochemica et Biophysica Acta 1219 (1994) –Pyrophosphate –5’  3’ exonulease activity –Variation of pH with temperature

Related Work III DNA and Cell Biology 10 (1991) The specificity of and amplification of PCR from the viewpoint of annealing

Denaturation Melting curve 의 each point 를 equilibrium 상태로 가정 Melting curve 에서 바로 ssDNA 의 fraction 을 계산  denaturation efficiency

Annealing Competitive hybridization between primers and ssDNAs Hybridization –Meet and Zip –Meeting is rate determining step Assumptions –Equilibrium is shifted to double strand formation  consider only forward reactions –ssDNAs are consumed completely

Reversible reaction Assume irreversible reaction No existence of ssDNA after the annealing step Assume Watson and Crick strand are identical Kinetic constants: from references or experiments

Extension Assumptions –No primer-dimer formation –No non-specific product formation Considerations –Enzyme inactivation with time and temperature –Decrease of efficient enzyme concentration due to the product accumulation –Enzyme-independent/dependent extension phase Biotechnology and bioengineering (1997)