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Effects of Quantum Dots in Polymerase Chain Reaction Libing Wang,†,‡ Yingyue Zhu,‡ Yuan Jiang,§ Ruirui Qiao,| Shuifang Zhu,⊥ Wei Chen,*,‡ and Chuanlai Xu*,‡ Hunan Import and Export Inspection and Quarantine Bureau, Changsha, HuNan, 410000, PRC, School of Food Science & Technology, Jiangnan UniVersity, Wuxi Jiangsu, 214122, PRC, Food Laboratory, Jiangsu Import and Export Inspection and Quarantine Bureau, Nanjing, JiangSu, 210000, PRC, Institute of Chemistry, CAS, Zhong Guan Cun, Bei Yi Jie 2, Beijing 100081, PRC, and Chinese Academy of Inspection and Quarantine, Beijing 100082, PRC ReceiVed: March 15, 2009; ReVised Manuscript ReceiVed: April 11, 2009
The effects of quantum dots (QDs) on the elimination of nonspecific amplification of the polymerase chain reaction (PCR) were investigated. It was found that QDs could increase the specificity of the PCR at different annealing temperatures and with DNA templates of different lengths. The effects of QDs on the efficiency of the PCR were also studied, and the results showed that there was no enhancement. The mechanisms underlying these effects are discussed. This method could be used to modify the amplification results of the conventional PCR. Furthermore, this technology could make the PCR more widely applicable, especially in the multi-PCR reaction system with different annealing temperatures. This is of great significance for gene diagnosis. Introduction Nanomaterials have been used in different fields with the rapid development of nanotechnology. Magnetic nanoparticles have been used in magnetic separation of biomolecules, targeted drug delivery, and cancer thermal therapy.1-4 Carbon nanotubes have been used to fabricate electronic devices and used as optic spectrum research targets.5-7 Mesporous silica have been used as catalysts and medicines and nucleic acid controlled release vectors.8-11 Gold nanoparticles have been used as transmission electron microscopy stain materials and cancer therapy.12 Also, gold nanoparticles are used as the blocks in the self-assembly materials and used in polymerase chain reaction (PCR) systems which take the different effects such as the nonspecific amplification optimal factor and amplification efficiency enhancement factor.13-18 PCR, arguably one of the most important inventions in the past decades, has found widespread use in the biological and medical sciences. Owing to its exponential amplification ability, this highly sensitive technique enables a broad spectrum of applications, including DNA sequencing, molecular diagnosis, and genetic analysis, even from a single pair of DNA molecules.17-26 Nevertheless, although it is highly sensitive, the current PCR technique is limited by its relatively low level of specificity. To reduce nonspecific amplification, systematic optimization of the operating conditions is generally required, such as the type and concentration of the enzyme, salt concentration, time and temperature of the annealing process, and primer design.27 It was discovered recently that the use of Au nanoparticles can reduce the level of nonspecific amplification at low temperature.17,18 This increased amplification specificity is believed to be due to enhanced heat transfer, as well as the effective binding of single-stranded DNA (ssDNA) to the * Corresponding author. E-mail:
[email protected], (W.C.);
[email protected], (C.X.). † Hunan Import and Export Inspection and Quarantine Bureau. ‡ Jiangnan Univsersity. § Jiangsu Import and Export Inspection and Quarantine Bureau. | CAS. ⊥ Chinese Academy of Inspection and Quarantine.
nanoparticles in a manner analogous to that of the ssDNAbinding protein SSB. This discovery opens a new avenue toward better amplification specificity through using other lowdimensional materials, such as quantum dots (QDs), nanorods, and nanowires, as PCR-assisting agents. We report, for the first time, the use of CdTe QDs as a novel assisting agent to increase the level of specificity of the amplification in the PCR technique. QDs, as a new kind of fluorescent material, possess many excellent characteristics, such as size-tunable emission, wide absorbance bands, narrow symmetric emission bands, high photostability, etc. QDs and Au nanoparticles enhance the specificity of the PCR, but QDs have the added advantage of fluorescence at different wavelengths and could serve as potential fluorescent materials in realtime PCR. In this work, carboxyl-based QDs with emission wavelengths in the range 530-630 nm were used as the model system. Systematic studies were undertaken to quantify the effects of QD under the conditions of different concentrations, annealing temperatures, and lengths of the DNA template on the specificity of the amplification and the efficiency of the PCR. Our results suggest that the use of QDs at the appropriate concentration can indeed improve the level of specificity of the PCR, even at a low annealing temperature and with a long DNA template. Experimental Section Gold nanoparticles (13 nm diameter) were synthesized as described.28 The thiolglycolic acid modified CdTe quantum dots with a tunable diameter in the range 2-10 nm and an emission spectrum from 530 to 630 nm (see Supporting Information Figure S1) were prepared as described.29,30 λ DNA (Dalian TaKaRa Bio. Inc., China) was used as the PCR template. Primers were purchased from Shanghai Sangon Biological Engineering Technology & Services Co. Ltd., China, including the forward primer 5′-GCAGTTGCCGTTTATCTCACC-3′ and the reverse primer 5′-GGCATAGCGTCCTCACATTTC-3′ for amplification of the 297 base template. Other primers for amplification of templates of other lengths were synthesized by the same company, and the sequences are given in Table 1.
10.1021/jp902404y CCC: $40.75 2009 American Chemical Society Published on Web 05/01/2009
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TABLE 1: Primer Sequence of Different Templates with Different Lengths primer name 297bp-F 297bp-R 594bp-F 594bp-R 1003bp-F 1003bp-R 8000bp-F 8000bp-R
primer sequence 5′-GCAGTTGCCGTTTATCTCACC-3′ 5′-GGCATAGCGTCCTCACATTTC-3′ 5′-TGAGCGGATACGGCGTGAAC-3′ 5′-CGCGACCAGTCAACGTCTGA-3′ 5′-GGCCATAAAAGGTCTTGAGC-3′ 5′-CGGCATTGTAGGATTTGGTA-3′ 5′-GATGAGTTCGTGTCCGTACAAC-3′ 5′-GACAATCTGGAATACGCCACC-3′
The PCR reaction was performed with 30 µL of reaction buffer (50 M KCl, 10 mM Tris-HCl pH 9.0 at 25 °C, 0.1% Triton X-100, 1.5 mM MgCl2), the four dNTPs at 2 mM each, 1 µL (5 ng) of pSK plasmid, and 0.5 U of Taq DNA polymerase. Two primers (100 pmol of each) were added to the PCR system. PCR was done with the Eppendorf PCR system (Eppendorf, U.S.), using the following protocol: 2 min at 94 °C; 35 cycles of 20 s denaturing at 94 °C, 20 s annealing at 55 °C, 45 s extension at 72 °C; and finally 3 min at 72 °C. Only the annealing temperature was changed for the experiments at different annealing temperatures. The PCR products were collected and submitted to electrophoresis in TBE/1.2% agarose gel. Real-time PCR was used to determine the efficiency of the PCR as follows: 40 s at 95 °C; 35 cycles of 20 s at 95 °C, 20 s at 55 °C, 45 s at 72 °C; 1 s at 95 °C, 15 s at 45 °C; and finally 30 s at 40 °C. Results and Discussion Effect of Quantum Dot Concentration. Parallel PCR procedures were done at an annealing temperature of 55 °C to examine the feasibility of using QDs to enhance the specificity of amplification in the PCR. Figure 1 shows the agarose gel electrophoresis of the corresponding PCR products. Compared with lane 8 (marker), lane 6 (control sample without QDs or Au nanoparticles) shows a broad distribution of amplified products, indicating nonspecific amplification. It has been reported that the addition of Au nanoparticles may effectively eliminate the nonspecific products.17,18 Lanes 1-4 show the amplified products in the presence of 0.01, 0.1, 0.4, and 0.6 nM Au nanoparticles, respectively. It is obvious that an increase of the concentration of the Au nanoparticles favors the elimina-
Figure 1. Effect of QDs on the specificity of PCR. The PCR was performed by employing a 297 bp target sequence from a λ DNA template, and PCR products were analyzed by agarose gel electrophoresis (1.5%). Lane 8, marker; lane 6, the control without nanomaterials; lane 7, the blank water control. Lanes 1-4 contain the gold nanoparticle: lane 1, 0.01 nM; lane 2, 0.1 nM; lane 3, 0.4 nM; lane 4, 0.6 nM. Lanes 9-12 contain the QDs with the 630 nm wavelength: lane 9, 0.25 nM; lane 10, 2 nM; lane 11, 4 nM; lane 12, 10 nM.
Figure 2. Elimination effect of the QDs with the 545 nm, 630 nm wavelength and the Au nanoparticle at different annealing temperature PCR systems: (A) 30 °C; (B) 35 °C; (C) 40 °C; (D) 45 °C. The PCR was performed by employing a 297 bp target sequence from a λ DNA template, and PCR products were analyzed by agarose gel electrophoresis (1.5%). In Figure 2A-D, lanes 1, 3, 5, and 8 were the controls without nanomaterials, lane 2 contained gold nanoparticle 0.4 nM, lane 4 contained QD 630 4 nM, lane 9 contained QD 545 13 nM, lane 6 was the blank water control, and lane 7 was the marker.
tion of the nonspecific amplified product. However, a high concentration of nanoparticles may inhibit the formation of
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Figure 4. Efficiency experiment of the QDs and gold nanoparticles. Lane 1, 1/5 time of normal PCR; lane 2, 1/10 time of normal PCR. Lanes 3 and 4 contained gold nanoparticles 0.4 and 0.6 nM, respectively. Lanes 5 and 6 contained the QDs 630 nm 4 and 6 nM, respectively.
Figure 3. Elimination effects of the QDs with different length templates in PCR: (A) 594 bp; (B) 1003 bp; (C) 8000 bp. Lanes 1, 3, 5, and 8 were the controls without nanomaterials, lane 2 contained gold nanoparticle 0.4 nM, lane 4 contained QD 630 4 nM, lane 9 contained QD 545 13 nM, lane 6 was the blank water control, and lane 7 was the marker.
specific products, as shown by the weakness of the specific product band in lane 4, which is consistent with earlier reports.17,18,31 Lanes 9-12 show the PCR products in the presence of QDs (630 nm emission wavelength) at a concentration of 0.25, 2, 4, and 10 nM, respectively. QDs and Au nanoparticles show a similar amplified effect except for QDs at a higher concentration. This study suggests the feasibility of using QDs for multiple PCR reactions with enhanced specificity. Note that an appropriate concentration of QDs may improve the level of specificity significantly, but an excessive concentration may inhibit the amplification process; therefore, 4 nM QD were chosen for the remaining studies. Effect of Annealing Temperature. The annealing temperature is an essential and important factor that may affect the specificity and efficiency of the PCR. Usually, a higher annealing temperature will reduce the efficiency of the PCR and a lower annealing temperature will induce nonspecific amplification. In this study, PCR was done with different annealing temperatures in the presence of QDs or Au nanoparticles. Figure 2 shows agarose gel electrophoresis of the PCR
products using an annealing temperature of 30 °C; lane 7 is a marker, and lanes 1, 3, and 5 are control samples (without QDs or Au nanoparticles) showing displacement of the bands of nonspecific amplified products. The addition of 4 nM QDs with an emission wavelength of 630 nm (lane 4) or 4 nM QDs with an emission wavelength of 545 nm (lane 9) eliminates the nonspecific bands, similar to what is seen with the addition of 0.4 nM Au nanoparticles (lane 2). The experiments done at 35, 40, and 45 °C gave similar results (Figure 2). The results of this study suggest the possibility of using QDs as a specificityenhancing agent for an error-prone PCR process, particularly at low annealing temperatures, and perhaps more importantly, this technology is shown to be a potentially useful tool for the multi-PCR system. Effect of Amplification Template Length. In theory, the PCR technique may be used to amplify a template of any length, but it is well-known that the PCR process with a long sequence template is prone to making mistakes. In practice, template lengths in the range of 100-1000 bases are commonly used. The longer the amplified template, the more extension time is needed, which will inevitably induce nonspecific amplification. We analyzed further whether QDs were suitable for PCR amplification with long templates to give large products. Figure 3 shows the results of the PCR amplification of sequence templates of 593, 1003, and 8000 bp. Both nonspecific and specific products were obtained with the standard PCR (Figure 3). However, only specific products were amplified when QDs were added to the PCR system with the 297, 593, or 1024 bp template. When the 8000 bp template was used, the elimination of the nonspecific products of the PCR was not as obvious as it was with the shorter sequence templates, even when QDs were added. There were some nonspecific tailing bands even after this optimization of the PCR, but it should be noted that nonspecific products both larger and smaller than the specific products were eliminated; i.e., there was no bias toward the smaller products. PCR Efficiency. Agarose electrophoresis and real-time PCR were used to confirm whether or not the addition of QDs enhanced the efficiency of the PCR. Figure 4 shows that the yield of the PCR product was reduced when the length of time of the cycle step of the standard PCR protocol was reduced. QDs and Au nanoparticles at the optimized concentration were included in the time-shortened PCR system, and the yield of the PCR product was characterized by TBE/agarose electrophoresis. Figure 4, lanes 1 and 2, shows the PCR result using 1 /5 and 1/10 of the amplification time of the standard PCR,
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Figure 5. Efficiency enhancement result by the real-time PCR.
respectively. There is clear linear correlation between the increased yield of the PCR product and the application time. Lanes 3 and 4 show the result with Au nanoparticles added using 1/5 and 1/10 of the amplification time of the standard PCR, respectively, and lanes 5 and 6 show the result with the QDs (630 nm emission wavelength) added using 1/5 and 1/10 of the amplification time of the standard PCR, respectively. Unfortunately, neither the QDs nor the Au nanoparticles showed any enhancement of the efficiency of the PCR. Conversely, the existing bands in lanes 1 and 2 disappeared in lanes 3-6. The result was further confirmed by the real-time PCR. As shown in Figure 5, there was no obvious enhancement of the efficiency of the PCR after adding QDs or Au nanoparticles at different concentrations. This result was different from that reported by Li18 but in accordance with the work by Vu at al.31 According to the reported theory, QDs are semiconductor nanoparticles composed of nonmetal elements, and the thermal conductivity is not as good as that of the metallic nanocolloids. This may be the reason why the QDs did not enhance the efficiency of the PCR. There are two possible reasons why the QDs do enhance the specificity of the PCR. From one aspect, we could think the optimization effect of the QDs to the specificity of the PCR is attributed to the similar optimization mechanism of the singlestranded binding protein (SSB), which selectively binds to the single-stranded DNA rather than double-stranded DNA and then minimizes the misparing between the primers and the templates in the PCR system.32-34 Why do the QDs have a similar optimization mechanism as the SSB in the PCR? First, the surface of the QDs used in this study was modified with carboxyl groups, which is responsible for the negatively charged surface of the QDs. Double-stranded DNA (dsDNA) has a higher surface charge density and is more repulsive than ssDNA in the negative atmosphere. Thus, the negatively charged QDs bind more easily to the anionic ssDNA strands than to dsDNA,35 which is similar to the SSB protein that binds to ssDNA selectively. Second, the rigidity of dsDNA does not favor the wrapping of the dsDNA around the QDs, while ssDNA is a soft, flexible polymer with a much greater degree of freedom
to wrap around the QDs. This selectivity greatly minimizes mispairing between primers and template during DNA replication, which is also similar to the SSB. From the other aspect, the optimization effect of the QDs may be due to the affinity between the DNA polymerase and the QDs. The DNA polymerase could be adsorbed onto the QDs, which would cause a reduction of the effective concentration of the polymerase in the PCR system. Therefore, under these conditions, only the target PCR product, which was annealed with primers most efficiently, would be amplified preferentially. As more QDs were added, more polymerase was adsorbed. When adequate QDs were added into the PCR system, the concentration of polymerase was decreased to less than the optimal effective concentration for specific amplification. That was why we found that the specific products were also eliminated when adequate QDs are added. Further experiments about this are in progress in our group. Conclusion The synthesized different wavelength QDs did indeed dramatically improve the amplification specificity of the PCR. In the error-prone PCR system, the specific amplification products were achieved when the different QDs were added into the PCR system. The optimal concentration of the added QDs was analyzed carefully. Furthermore, the QDs could also be used in the long sequence template amplification. Only the specific target templates were amplified with the adding of the QDs in the optimal concentration. On the basis of the research results, the QDs could be used as the optimizing factor in the conventional PCR, especially multiplex PCR, in which multiPCR amplification with different anneal temperatures could be carried out in the same system at the same time. QDs could be used to improve the PCR results under certain conditions by applying this novel method. In conclusion, the QDs are of great importance for the wide application of the PCR. Acknowledgment. We acknowledge the financial support provided by the National Natural Science Foundation of
Effects of Quantum Dots in Polymerase Chain Reaction China (20675035, 20871060, 20835006), 11th Five Years Key Programs for Science and Technology Development of China (2006BAK02A09, 2006BAK02A19, 2006BAK02A06, 2006BAK10B04, 2007BAK26B06, 200810099, 200810219, 2006AA10Z450, 2006BAD27B02, 2006BAF07B01, 2006BAD04A08, 2008ZX0812, and 2006BAK02A29) and supported by 111 project-B07029. Supporting Information Available: Figure showing the fluorescent image and spectrum of the different QDs. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Engel, H.; Kleespies, C.; Friedrich, J.; Breidenbach, M.; Kallenborn, A.; Schondorf, T.; Kolhagen, H.; Mallmann, P. Br. J. Cancer 1999, 81, 1165. (2) Ito, A.; Matsuoka, F.; Honda, H.; Kobayashi, T. Cancer Immunol. Immunother. 2004, 53, 26. (3) Wada, S.; Tazawa, K.; Furuta, I.; Nagae, H. Oral Dis. 2003, 9, 218. (4) Alexiou, C.; Jurgons, R.; Seliger, C.; Iro, H. J. Nanosci. Nanotechnol. 2006, 6, 2762. (5) Schipper, M. L.; Ratchford, N. N.; Davis, C. R.; Kam, N. W. S.; Chu, P.; Liu, Z.; Sun, X. M.; Dai, H. J.; Gambhir, S. S. Nat. Nanotechnol. 2008, 3, 216. (6) Welsher, K.; Liu, Z.; Daranciang, D.; Dai, H. J. Nano Lett. 2008, 8, 586. (7) Zhang, L.; Zaric, S.; Tu, X. M.; Wang, X. R.; Zhao, W.; Dai, H. J. J. Am. Chem. Soc. 2008, 130, 2686. (8) You, Y. Z.; Kalebaila, K. K.; Oupicky, D. Chem. Mater. 2008, 20, 3354. (9) Lu, Y. F. Angew. Chem., Int. Ed. 2006, 45, 7664. (10) Ji, X.; Hu, Q.; Hampsey, J. E.; Lu, Y. F. Chem. Mater. 2006, 18, 2265. (11) Peng, H.; Tang, J.; Yang, L.; Pang, J.; Ashbaugh, H. S.; Brinker, C. J.; Yang, Z.; Lu, Y. F. J. Am. Chem. Soc. 2006, 128, 5304. (12) Qian, X.; Peng, X. H.; Ansari, D. O.; Yin-Goen, Q.; Chen, G. Z.; Shin, D. M.; Yang, L.; Young, A. N.; Wang, M. D.; Nie, S. M. Nat. Biotechnol. 2008, 26, 83.
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