Variability in DNA Polymerase Efficiency: Effects of Random Error, DNA Extraction Method and Isolate Type
نویسنده
چکیده
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%).
منابع مشابه
A Simple and Rapid Leaf Genomic DNA Extraction Method for Polymerase Chain Reaction Analysis
In plants, secondary metabolites and polysaccharides interfere with genomic isolation procedures and downstream reactions such as restriction enzyme analysis and gene amplification. The removal of such contaminants needs complicated and time-consuming protocols. In this study, a simple, rapid and efficient method for leaf DNA extraction was optimized. This method use small amount of plant mater...
متن کاملThe Comparison of different Procedures for DNA extraction from paraffin-embedded Tissues: A commercial kit and a traditional method based on heating
Abstract Background and objectives: Paraffin-embedded tissues and clinical samples are a valuable resource for molecular genetic studies, but the extraction of high-quality genomic DNA from this tissues is still a problematic issue. In the Present study, the efficiency of two DNA extraction protocols, a commercial kit and a traditional method based on heating and K Proteinase was compared. Mate...
متن کاملEvaluation of a modified salt-out method for DNA extraction from whole blood lymphocytes: A simple and economical method for gene polymorphism
Extraction of high-quality and-quantity DNA is a fundamental requirement for genetic research. It is very important to address the use of DNA extraction methods that are simple and cost-effective in gene polymorphism with large number of samples. This study was designed to investigate the optimal DNA extraction from lymphocytic cells by salt-out method. In this study, 200 blood samples of the t...
متن کاملA RAPID AND SIMPLE METHOD OF DNA EXTRACTION FROM CLINICAL SPECIMENS CONTAINING MYCOBACTERIUM LEPRAE FOR PCR ASSAYS
We describe a rapid, simple, and reliable procedure for routine isolation of Mycobacterium leprae DNA from slit-skin swab specimens. This one-step DNA extraction method is based on the utilization of ChelexR 100, a chelating ion exchange resin. Slit-skin swab specimens from seven untreated leprosy patients at Baba Baghi Leprosy Hospital in Iran were processed by this procedure. The polymer...
متن کاملRapid DNA extraction of bacterial genome of Staphylococcus aureus using laundry detergents and assessment of the efficiency of DNA in downstream process using PCR
Abstract Background and objectives: Genomic DNA extraction of bacterial cells is of processes performed normally in most biological laboratories therefore, various methods have been offered, manually and kit, which may be time consuming and costly. In this paper, genomic DNA extraction of Staphylococcus aureus was investigated using some laundry detergent brands available in Iran to achieve ...
متن کامل