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

An investigation of the influence of five DNA polymerase-buffersystems on real-time PCR showed that the choice of both DNApolymerase and the buffer system affected the amplificationefficiency as well as the detection window. The analytical repeatabilityof the data for different systems changed clearly, leading usto conclude that basing quantitative measurements on single-data-setstandard curves can lead to significant errors.

INTRODUCTION

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Introduction

Sequence-specific nucleic acid quantification in areas suchas diagnostic PCR and molecular biology has been greatly improvedby the introduction of real-time PCR technology (9). While thistechnology has tremendous potential for accurate and sensitivequantification, further studies addressing the quantificationaspect of this technology are required before it can be widelyimplemented. Previous results from this laboratory (5), in whichthe range of detection of real-time PCR was modeled in a puresystem using two different DNA polymerases, gave an indicationthat DNA polymerases and their buffer systems influence theperformance of PCR by affecting the detection window and linearrange of amplification. The aim of this work was to systematicallystudy the effect of five DNA polymerase-buffer systems on absolutequantification 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 with4 mM MgCl₂. Sterile Millipore water was added to a volume of16 µl and complemented with 4 µl of Y. enterocoliticaDNA. The concentration of DNA was fluorimetrically determinedusing a TD-700 fluorimeter (Turner Designs, Sunnyvale, Calif.),and the DNA was diluted to appropriate concentrations in sterileMillipore water. The four other master mixtures, contained 2.5U of DNA polymerase and 1x associated buffer, 4 mM MgCl₂, 0.4ml of each primer, 0.2 mM (each) deoxynucleoside triphosphate,10,000-fold-diluted SYBR Green I, and 4 µl of Y. enterocoliticaDNA in a total volume of 20 µl. The following DNA polymeraseswere used: DyNazyme II (FINNZYMES OY, Espoo, Finland), rTth(Applied Biosystems, Foster City, Calif.), and Taq (Roche Diagnostics)and Tth (Roche Diagnostics). Each amplification started witha denaturation step of 1 min at 95°C, followed by 40 cyclesof 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 fluorescencemeasurement and finally 25 s of final elongation. Amplificationwas followed by melting curve analysis between 65 and 95°Cand finally cooling for 1 min at 40°C. The quantificationdata, in terms of the crossing point value (ROM) (which is expressedas the fractional cycle number and is the intersection of thelog-linear fluorescence curve with a threshold crossing line),were determined using the second derivative method of the LightCyclerSoftware, version 3 (Roche Diagnostics).

Amplification efficiency and analytical repeatability.

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Amplification efficiency and...

Independent triplicates of 10-fold dilutions of Y. enterocoliticaDNA, from 1 mg/ml to 1 fg/ml, were used to obtain standard curvesfor each polymerase-buffer system (Fig. 1). After amplification,results from the melting curve were analyzed, and the ROM valuesof all samples that gave a positive specific product peak between88 and 92°C were plotted against the log of the initialDNA concentration. From this slope the amplification efficiencywas calculated using the equation E = (10^-1/slope) - 1 (4).Figure 1A to E shows the slope through all generated data, whileFig. 1F to J shows independent analysis of the triplicates foreach DNA polymerase. Assuming that all slopes should be -3.32(which would lead to the optimal amplification efficiency of1), differences can be seen between different DNA polymerasesin Fig. 1A to E (P = 0.053). In particular, Taq, Tth, and DyNazymeII have amplification efficiencies very close to 1. When lookingat Fig. 1F to J, it is clear that the different DNA polymerasesshow differences in repeatability, as this is defined as theintralaboratory variability. In particular, Taq and LCTaq showgreat variation between different runs.

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FIG. 1. Standard curves generated for five different DNA polymerase-buffer systems after two different types of analysis of the same independent triplicate data. Panels A to E show standard curves and corresponding equations when all three sets of data for each DNA polymerase were used for one analysis of DyNazyme II (A), LCTaq (B), rTth (C), Taq (D), and Tth (E). Panels F to J show standard curves and corresponding equations when the three sets of data were used for independent analysis of DyNazyme II (F), LCTaq (G), rTth (H), Taq (I), and Tth (J).

Detection window and detection probability.

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Detection window and detection...

From the triplicate analysis it is possible to create a detectionprobability graph showing the number of detectable points ateach DNA concentration (Fig. 2). A significant difference canbe seen between the results from the different DNA polymerase-buffersystems. Thus, the detection window for Tth is the broadest,with at least 67% detection probability over a window of 8 logunits, compared to Taq and LCTaq, with a window of 6 log units.This confirms previous indications about a possible differencebetween the performance of different DNA polymerase-buffer systemsin real-time PCR, as proposed by Knutsson et al. (5). The resultsfor both amplification efficiency and detection window wereconfirmed by repeating this test with another primer pair, codingfor a 0.6-kb region of the Yersinia virulence gene yadA (6),which showed the same trends (data not shown). However, otherfactors, such as the thermal cycler model and the probe system,may affect the PCR per- formance.

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FIG. 2. Detection probability using different DNA polymerase-buffer systems. The detection probability for each DNA concentration was determined by checking the amount of data points/total amount of analysis. Data were determined after independent triplicate experiments. —•—, DyNazyme II; -- ${square}$ --, LCTaq; — ${blacktriangleup}$ —, rTth; ——, Taq; and ---x: Tth.

Impact on nucleic acid quantification.

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Impact on nucleic acid...

The effect of the DNA polymerase-buffer system on DNA quantificationwas demonstrated by quantifying four standardized DNA samples(Table 1). In particular, Taq and LCTaq generated less-accuratequantification data. The main reason for this is the narrowerdetection window and the greater deviations in the standardcurve.

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TABLE 1. The effect of the DNA polymerase and the accompanying buffer on quantitative real-time PCR

The effect of buffer composition.

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The effect of buffer...

The influence of buffer components on the amplification efficiencyand the detection window was determined for Taq and Tth (Table2). From the data it is clear that at least for Tth, the buffercomposition affects the detection window, since with increasingcomplexity of the buffer the detection window becomes wider.However, comparing data between Taq and Tth, it can be seenfor all buffers that the use of Tth improves the PCR performance.This implies that both buffer composition and DNA polymerasecan influence the results.

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TABLE 2. The influence of buffer components on amplification efficiency and detection window using Taq polymerase and Tth DNA polymerase

Correct data analysis.

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Correct data analysis.

There seems to be no consensus on the correct way to analyzequantitative data and to create standard curves for absolutequantification with real-time PCR. Most published data showstandard curves constructed from one data set (2, 7, 8), whileothers analyze and use multiple data sets to calculate the amplificationefficiency (1, 3). The data shown in Fig. 1 indicate that especiallywhen using certain DNA polymerases, such as Taq polymerase orrTth, the intralaboratory variation can differ between datasets and that it is of great importance to perform multipleanalyses. Furthermore, the linear range of amplification, thearea of the detection window in which a linear relationshipis obtained between the log DNA concentration and the Cp value,does not always seem to match the detection window. For example,the lowest DNA concentration for LCTaq seems to have such agreat variation between the points that it may be questionedwhether these data points should be used to calculate the amplificationefficiency. In conclusion, this study has shown that the DNApolymerase-buffer system used for quantitative analysis canimpact the performance of the system, and when used to quantifyunknown samples it affects the accuracy of the data. Furthermore,it has indicated a need for consensus on the correct way toanalyze quantitative PCR data in order to be able to comparethe performance of different assays.

ACKNOWLEDGMENTS

This work was financially supported by the Commission of theEuropean Communities within the program "FOOD-PCR," QLK1-1999-00226,and the Swedish Research Council for the Environment, AgriculturalSciences and Spatial Planning, 2001-4068.

FOOTNOTES

* Corresponding author. Mailing address: Applied Microbiology, Lund Institute of Technology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden. Phone: 46 46-222 3412. Fax: 46 46-222 4203. E-mail: peter.radstrom{at}tmb.lth.se.

REFERENCES

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