Accurate Estimation of Nucleic Acids by Amplification Efficiency Dependent PCR

<div><p>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 <i>E. coli</i>. 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.</p></div

Graphical representation of the eqn. logΔRn = nlogF + log (N₀/c).

<p>A straight line with a slope of logF and an intercept of N₀/c on the Y-axis is obtained by plotting LogΔRn versus n. From the slope logF, the amplification efficiency of each individual PCR reaction can be calculated.</p

Theoretically predicted PCR profile with an initial template concentration of 1 pg/100 ul and fragment length of 1000 bp.

<p>Theoretically predicted PCR profile with an initial template concentration of 1 pg/100 ul and fragment length of 1000 bp.</p

Theoretically predicted variation of amplification factor with number of cycles in an experiment where the initial template concentration is 1 pg/100 ul and length of the template 100 bp.

<p>Theoretically predicted variation of amplification factor with number of cycles in an experiment where the initial template concentration is 1 pg/100 ul and length of the template 100 bp.</p

Flow chart of the analysis used in RARE.

<p>Flow chart of the analysis used in RARE.</p

Relative levels of mRNA expression in TCA cycle/Glyoxylate Shunt pathway under acetate and glucose.

<p>Relative levels of mRNA expression in TCA cycle/Glyoxylate Shunt pathway under acetate and glucose.</p

Amplification efficiency determined by RARE for TCA cycle/Glyoxylate Shunt genes expressed under alternate carbon sources.

<p>Mean amplification efficiency and standard deviation were calculated from three identical PCR reactions for either acetate (white bar) or glucose (grey bar) and plotted against the different genes under study.</p

Correlation profile between the actual and the calculated initial template concentrations.

<p>Correlation profile between the actual and the calculated initial template concentrations.</p

Sequence of the 16 <i>E. coli</i> gene specific forward and reverse primers.

<p>Sequence of the 16 <i>E. coli</i> gene specific forward and reverse primers.</p

Purification of DHK and FabG.

<p><b>A</b>. SDS-PAGE analysis of the purified DHK. Lane 1: Purified DHK through Ni-NTA, Lane 2: Puregene Broad range marker, Lane 3: Superdex S75 purified DHK. <b>B.</b> SDS-PAGE of purified FabG Lane1: Low range marker Lane 2,3,4: gel purified FabG.</p