Impact of DNA Polymerases and Their Buffer Systems on Quantitative Real-Time PCR

Abstract

An investigation of the influence of five DNA polymerase-buffer systems on real-time PCR showed that the choice of both DNA polymerase and the buffer system affected the amplification efficiency as well as the detection window. The analytical repeatability of the data for different systems changed clearly, leading us to conclude that basing quantitative measurements on single-data-set standard curves can lead to significant errors.Sequence-specific nucleic acid quantification in areas such as diagnostic PCR and molecular biology has been greatly improved by the introduction of real-time PCR technology (9). While this technology has tremendous potential for accurate and sensitive quantification, further studies addressing the quantification aspect of this technology are required before it can be widely implemented. Previous results from this laboratory (5), in which the range of detection of real-time PCR was modeled in a pure system using two different DNA polymerases, gave an indication that DNA polymerases and their buffer systems influence the performance of PCR by affecting the detection window and linear range of amplification. The aim of this work was to systematically study the effect of five DNA polymerase-buffer systems on absolute quantification using the LightCycler instrument (Roche Diagnostics, Mannheim, Germany).A primer set, Y1 and Y2, for Yersinia enterocolitica was used (6). To a commercial LightCycler kit (LCTaq) (Roche Diagnostics), a 0.4 mM concentration of each primer was added together with 4 mM MgCl 2 . Sterile Millipore water was added to a volume of 16 l and complemented with 4 l of Y. enterocolitica DNA. The concentration of DNA was fluorimetrically determined using a TD-700 fluorimeter (Turner Designs, Sunnyvale, Calif.), and the DNA was diluted to appropriate concentrations in sterile Millipore water. The four other master mixtures, contained 2.5 U of DNA polymerase and 1ϫ associated buffer, 4 mM MgCl 2 , 0.4 ml of each primer, 0.2 mM (each) deoxynucleoside triphosphate, 10,000-fold-diluted SYBR Green I, and 4 l of Y. enterocolitica DNA in a total volume of 20 l. The following DNA polymerases were used: DyNazyme II (FINNZYMES OY, Espoo, Finland), rTth (Applied Biosystems, Foster City, Calif.), and Taq (Roche Diagnostics) and Tth (Roche Diagnostics). Each amplification started with a denaturation step of 1 min at 95°C, followed by 40 cycles of 0.1 s of denaturation at 95°C, 5 s of annealing at 60°C, and elongation for 15 s at 72°C, followed by a single fluorescence measurement and finally 25 s of final elongation. Amplification was followed by melting curve analysis between 65 and 95°C and finally cooling for 1 min at 40°C. The quantification data, in terms of the crossing point value (ROM) (which is expressed as the fractional cycle number and is the intersection of the log-linear fluorescence curve with a threshold crossing line), were determined using the second derivative method of the LightCycler Software, version 3 (Roche Diagnostics).Amplification efficiency and analytical repea...

Synergy between DNA polymerases increases polymerase chain reaction inhibitor tolerance in forensic DNA analysis

“…Buffer components may also be important in the sense that compounds present in the buffer system of one DNA polymerase may be beneficial for the activity of the other polymerase in the blend. The presence or absence of PCR facilitators is a well-known factor determining the amplification efficiency of DNA polymerases in general [35], and several different compounds have been used to counteract PCR inhibition [25]. Examples of PCR facilitators that have been shown to increase inhibitor tolerance include proteins such as BSA [14,26] and nonionic detergents such as Tween 20 and Triton X-100 [33,36].…”

Rapid Quantification ofYersinia enterocoliticain Pork Samples by a Novel Sample Preparation Method, Flotation, Prior to Real-Time PCR

“…In a previous study, it was shown that qPCR can be improved by the use of alternative DNA polymerases, such as Tth (28). By using this DNA polymerase and its buffer in flotationqPCR, quantification was possible over a range of at least 5 log units (Fig.…”

“…Real-time PCR conditions. A primer set targeting a 0.3-kb part of the 16S rRNA gene from Y. enterocolitica (16) was used to develop a real-time PCR assay using the LightCycler instrument (Roche Diagnostics, Mannheim, Germany) (28). The PCR mixture consisted of 0.75 U of Tth DNA polymerase (Roche Diagnostics); 1ϫ Tth DNA polymerase buffer (Roche Diagnostics); 4 mM MgCl 2 ; 0.4 M each primer; 0.2 mM (each) dATP, dCTP, dGTP, and dTTP; 10,000-fold-diluted SYBR Green I (Roche Diagnostics); and 4 l of sample.…”

Multi-platform comparison of ten commercial master mixes for probe-based real-time polymerase chain reaction detection of bioterrorism threat agents for surge preparedness

“…Their study [16] used one signature set and examined the ability of certain PCR enzymes to amplify in the presence of common PCR-inhibiting components found in complex biological samples. Subsequently, Wolffs et al [17] investigated four DNA polymerases (Taq, DyNazyme, Tth and rTth) and two different buffer systems in real-time PCR and showed that the choice of both the type DNA polymerase and the buffer system affected amplification efficiency. In our study, we compared 10 commercial reagents by the use of real-time PCR assays for four different select agents in an effort to provide a thorough evaluation of the most popular kits that are currently on the market.…”