Accurate Estimation of Nucleic Acids by Amplification Efficiency Dependent PCR

Accurate estimation of template--DNA or RNA by real time PCR is dependent on the amplification efficiency (F) of the reaction. The analytical equation describing the kinetics of PCR that is influenced by template re-annealing is formulated. It predicts the gradual reduction of F--from its initial value of 2, leading to template saturation. From an experimental standpoint, due to the exponential nature of the reaction a minute change in F can lead to a large error in the estimation of the initial template concentration. On the basis of individual variation in the amplification efficiency we have formulated a simple mathematical model and an MS Excel based data analysis software that allows accurate and automated quantification of initial template concentration. This method which does not require any normalisation with housekeeping genes was validated by transcript profiling of the genes in the TCA/glyoxylate cycle of E. coli. Consistent with published reports, we observed a precise and specific induction of the glyoxylate shunt genes when the bacteria was shifted from a six carbon glucose media to a two carbon source like acetate.