Enhanced Specificity of Multiplex Polymerase Chain Reaction via CdTe Quantum Dots
© Liang et al. 2010
Received: 7 July 2010
Accepted: 10 September 2010
Published: 30 September 2010
Nanoparticles were recently reported to be able to improve both efficiency and specificity in polymerase chain reaction (PCR). Here, CdTe QDs were introduced into multi-PCR systems. It was found that an appropriate concentration of CdTe QDs could enhance the performance of multi-PCR by reducing the formation of nonspecific products in the complex system, but an excessive amount of CdTe QDs could suppress the PCR. The effects of QDs on PCR can be reversed by increasing the polymerase concentration or by adding bovine serum albumin (BSA). The mechanisms underlying these effects were also discussed. The results indicated that CdTe QDs could be used to optimize the amplification products of the PCR, especially in the multi-PCR system with different primers annealing temperatures, which is of great significance for molecular diagnosis.
The PCR has become one of the most popular tool for molecular biology and pathogen detection as a high sensitive technique [1–3], but it still requires careful optimization on reaction conditions in order to eliminate nonspecific products in most PCR experiments, especially in multiplex PCR (multi-PCR). Optimized parameters could include the type and concentration of the enzyme, reaction buffer content, time and temperature of the annealing/extension process, primer design and use of a rapid heating–cooling response PCR machine [4, 5]. It was reported that a variety of organic chemicals, including single-stranded DNA-binding proteins (SSBs) , amides and betaine , imidazole, tetramethylammonium chloride (TMAC) and TMAC derivatives [6–9], could act as accelerants in PCR mixture to increase yield and specificity.
As a novel material, nanoparticles (NPs) have many important applications in biology, for example in drug delivery, biomolecules separation, cancer thermal therapy, ultrasensitive biosensor and live cell imaging [10–15]. Recently, gold nanoparticles have been reported to be able to reduce the level of nonspecific products even at lower annealing temperature [9, 16, 17]. Li et al.  found that Au NPs can improve the efficiency of PCR and attributed the enhancement to the excellent heat conductivity of Au NPs. But some researchers reported some discriminating conclusions [17–19]. Yang et al.  evaluated the function of Au NPs in PCR systems and suggested that a complex interaction between Au NPs and native Taq DNA polymerase was accounted for the effects of Au NPs on PCR. Vuet et al.  demonstrated that AuNPs tended to favor smaller fragments of PCR products than larger ones owing to the different polymerase adsorptions. Possible reasons concerning these previous discrepant results might be referred to different nanoparticles used in terms of surface modification and some other different experimental conditions.
Quantum dots, as a new kind of fluorescent material, have many excellent features, such as size-tunable emission, broad excitation profiles and narrow/symmetric emission spectra, high photostability. The new generations of QDs have far-reaching potential for the study of intracellular processes at the single-molecule level, high-resolution cellular imaging, long-term in vivo observation of cell trafficking, tumor targeting and diagnostics . CdTe QDs have the advantage of fluorescence at different wavelengths and could be used as potential fluorescent materials in real-time PCR. Here, CdTe quantum dots (QDs) were employed as a novel assisting agent to systematically investigate the effects of nanoparticles on PCR. First, two-round PCR was used to amplify a 310-bp fragment from Triticum aestivum genomic DNA and then the performance of PCR via the QDs was evaluated by multi-PCR and real-time PCR. The results showed that CdTe QDs could reduce the formation of nonspecific products at an appropriate concentration, but inhibit the amplification when using an excessive amount of QDs in the multi-PCR mixture. The mechanisms underlying these effects were also discussed.
Materials and methods
Preparation of CdTe QDs
The Template DNA and Primers
Primers for 18S rDNA templates
Sequence (5' to 3')
The Conventional PCR and Real-Time PCR Systems
The ABI2720 PCR machine and the ABI 7500 real-time PCR System (Application Biosystems, Foster, USA) were used in this study. For specificity test, two-round PCR was used in which the PCR products from the first round were used as the DNA template for the second-round PCR [9, 22]. The specificity of amplification was also tested using multiplex PCR in which two reverse primers and two different forward primers were used. Each conventional PCR was carried out with 50 pg–15 ng of DNA template, 0.25 μM of each primer and 12.5 μl of BioTaq master mix (BioTeke, Beijing, China) in a final volume of 25 μl. As a parallel assay, rTaq DNA polymerase (TaKaRa, Dalian, China) was used in the study. PCR was run in an ABI 2720 cycler (applied Biosystems) with 35 cycles of 15 s denaturation at 95°C, 35 s annealing at 50°C, followed by 50 s extension at 72°C. Cycling was started after an initial denaturation at 95°C for 5 min and ended with a final extension at 72°C for 5 min. Only the annealing temperature was changed for the experiments at different annealing temperatures. The PCR products were collected and submitted to electrophoresis in 1.6%/TBE agarose gel.
ABI 7500 real-time PCR system and its SDS software (version 1.40) were used to evaluate amplification efficiency. The 20 μl reactions were carried out including 250 pg of DNA template, 0.2 μM of each primer and 1× SYBR® Green PCR master mix (TaKaRa, Dalian, China). The PCR temperature is as follows: 5 min at 95°C; 40 cycles of 15 s at 95°C, 1 min at 60°C.
Results and Discussion
Effects of CdTe QDs Concentrations on the Specificity of PCR
In previous experiment, another interesting phenomenon was observed (Figure 4). The specific bands 647 and 603 bp gradually disappeared with the increase in QDs concentration while that of the short target fragments of 153 and 109 bp become gradually brighter, and all bands disappeared in 100 nM lane. This indicates that the CdTe QDs could optimize the PCR process through modulating the concentration of the component in PCR mix.
Reverse Effects of CdTe QDs by BSA and Polymerase
Effect of QDs on PCR Efficiency
Why the QDs could improve the specificity of the PCR? There are two possible reasons for the question. On the one hand, the improvement effect of the QDs to the specificity of the PCR could be attributed to the similar optimization mechanism of the single-stranded binding protein (SSB), which selectively binds to the single-stranded DNA rather than double-stranded DNA and then minimizes the mispairing between the primers and the templates in the PCR system [6, 24]. On the other hand, the interaction between the DNA polymerase and the QDs might enhance the specificity of PCR. The possibility was confirmed by our experiments. The effects of CdTe QDs on PCR did not alter by increasing the concentration of template or primers, but were completely reversed by increasing the concentration of Taq polymerase in the PCR mixture. This suggested there was an interaction between QDs and DNA polymerase. Additionally, the effects of CdTe QDs on PCR can be reversed by adding BSA as a competitor with QDs in the reaction, strongly implying that the action of CdTe QDs in PCR might be through the adsorption and desorption of polymerase. The adsorption of polymerase by CdTe QDs would lead to a reduction in effective polymerase concentration. When polymerase concentration become rare limited, only the target PCR products, which were annealed with primers most efficiently, would be amplified preferentially. While PCR with excessive polymerase concentration tends to produce high molecular weight nonspecific products, and at lower polymerase concentrations might be anticipated to show lower processivity and bias toward smaller fragments . This was the possible reasons why the PCR products were shifted from the larger amplicon to the smaller amplicon observed in Figure 4. Finally, the specific products were also eliminated when adequate QDs are added into the reaction mixture. This phenomenon was validated with rTaq and power Taq polymerase in our study.
As a polypeptide molecule, DNA polymerase can be interacted with other biomolecules, such as protein, nucleic acid. The present results indicated that CdTe QDs could regulate the effective polymerase concentration in PCR process. Being different from other binding factors of protein, the binding process of CdTe QDs can be regulated and reversed by adding a competitor, i.e., BSA. The present experiments showed that low concentration of CdTe QDs had no obvious effect on the PCR process but excess CdTe QDs would result in the repression of PCR process, and only appropriate concentration of QDs could improve PCR specificity. Inspired by these results, we propose a possible mechanism that CdTe QDs optimize the PCR process by regulating the dynamic equilibrium between DNA polymerase and QDs. A central idea of the hypothesis was from the reversible binding of CdTe QDs and free polymerase. As well known, the polymerization is consisted of two processes in PCR system, i.e., the binding of polymerase with primer–template junction (PTJ) and extension of the binding. Higher concentration polymerase might have more opportunity to bind PTJ, which squint toward increase in the average length of products synthesized on a long template, result in more nonspecific products . By regulating the dynamic equilibrium of binding of polymerase with primer–temples junction, the PCR process and DNA synthesis could be modulated. Whereas low concentration CdTe QDs have no effect on the equilibrium and higher concentration CdTe QDs might inhibit the interaction of primer–template–polymerase. Considering that PCR is a very complex process, intensive studies should be carried out in order to reveal the underlying mechanism of QD-PCR.
As gold and other nanoparticles, the synthesized carboxyl-based CdTe QDs did indeed effectively improve the PCR specificity. In the normal PCR and multi-PCR system, the specific PCR products were achieved when the different concentrations of QDs were added into the PCR system. The optimal concentration of the added QDs was obtained in present multi-PCR systems. Based on the research results, the QDs could be used as a useful additive in the PCR, especially multiplex PCR. The ability of QDs to improve the specificity of PCR is advantageous for the expansion of its biological applications. Being the complexity of PCR process, intensive studies should be carried out in order to reveal the in-depth mechanism of QDs in multiplex PCR.
This work was financially supported by the National Basic Research Program of China (973 Program: 2007CB936300), NSFC (No. 20875014, No. 30870626) and 2008DFA51180.
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