Variability in DNA Polymerase Efficiency: Effects of Random Error, DNA Extraction Method and Isolate Type

Using computer-generated data calculated with set amounts of random error (E = 1, 5 & 10%) associated with calculated qPCR cycle number (C ) at four jth 1:10 DNA dilutions, we found that the “efficiency” (ε) associated with each population distribution of n = 10,000 measurements varied from 0.95 to 1.05 for E = 1% (ε average = 1.00 ± 0.0132; ε coefficient of variation or CV ~ 1%), 0.85 to 1.2 for E = 5% (ε average = 1.00 ± 0.0665; fraction of observed distribution between ε= 0.9 and 1.1 = 89%; ε CV ~ 7%), and 0.7 to 1.6 for E = 10% (ε average = 1.02 ± 0.139; fraction of distribution between ε = 0.9 and 1.1 = 54%; ε CV ~ 13%). The data associated with the highest error rate also displayed a large asymmetry in the ε frequency distribution whereupon the 3rd central moment was about 8-fold greater than the distribution linked to the lowest rate of error. Not surprisingly, this skewness was shown to be associated with the ε calculation since the distribution of ∂Cj/∂Log10[0.1] (10% error; j = 0, 1, 2, 3), from which ε is calculated, was normally distributed (Gaussian distribution function: μ = -3.32 ± 0.00399 and σ = 0.327 ± 0.00327; ± asymptotic standard error). To better identify potential sources of such variation, we investigated DNA standards of known concentration (n = 8 independent sets of experiments of 6 dilutions each) amplified from 3 isolates to test the typical threshold cycle number qPCR data (Ct) with that of the less used derivative method (C∂: interpolated value of C where the second derivative in normalized fluorescence with respect to C is equal to 0) and found that the average CV dropped from 4.35% to 2.70% using the latter method. Using this less error-prone variable (C∂), we tested 11 different DNA extraction technologies (applied to 1 Gram-positive and 2 Gram-negative organisms using primers specific for their 16S rRNA “genes”) and found that only 3 of these methods showed significant variation in ε due to extraction method alone (averaged across 3 replicates/isolate × 3 isolates). Applying 3 of the most effective (i.e., those with the greatest 16S rDNA copies per colony forming unit) DNA extraction methods to 5 different Grampositive and 10 Gram-negative organisms, only two organisms showed significant variation in ε due to organism alone (averaged across 3 replicates/method × 3 methods using a universal primer for the 16S rRNA “gene”). Overall, the implication is that most of the variation in qPCR ε is random (CV ~ 8%). However, certain DNA extraction methods and isolates did induce a greater variation in ε (CV~11%).