Facilitation of Polymerase Chain Reaction with Poly(ethylene glycol

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Facilitation of PCR with Polyethylene Glycol-Engrafted Graphene Oxide Analogous to Single-strand DNA-binding Protein Hyo Ryoung Kim, Ahruem Baek, Il Joon Lee, and Dong-Eun Kim ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b13223 • Publication Date (Web): 22 Nov 2016 Downloaded from http://pubs.acs.org on November 28, 2016

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ACS Applied Materials & Interfaces

Facilitation of PCR with Polyethylene GlycolEngrafted Graphene Oxide Analogous to Singlestrand DNA-binding Protein Hyo Ryoung Kim, Ahruem Baek, Il Joon Lee, and Dong-Eun Kim* Department of Bioscience and Biotechnology, Konkuk University Neundong-ro 120, Gwangjin-gu, Seoul 05029, Republic of Korea E-mail: [email protected]

KEYWORDS : Graphene oxide, nanomaterials, poly(ethylene glycol), polymerase chain reaction, single-stranded DNA

ABSTRACT: Polymerase chain reaction (PCR), a versatile DNA amplification method, is a fundamental technology in modern life sciences and molecular diagnostics. After multiple rounds of PCR, however, nonspecific DNA fragments are often produced and amplification efficiency and fidelity decrease. Here, we demonstrated that polyethylene glycol-engrafted nanosized graphene oxide (PEG-nGO) can significantly improve PCR specificity and efficiency.

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PEG-nGO allows specificity to be maintained even after multiple rounds of PCR, allowing reliable amplification at low annealing temperatures. PEG-nGO decreases nonspecific annealing of single-stranded DNA (ssDNA), such as primer dimerization and false priming, by adsorbing excess primers. Moreover, PEG-nGO interrupts reannealing of denatured template DNA by preferentially binding to ssDNA. Thus, PEG-nGO enhances PCR specificity by preferentially binding to ssDNA without inhibiting DNA polymerase, which is analogous to the role of singlestranded DNA binding proteins.

1. INTRODUCTION The polymerase chain reaction (PCR), an artificial DNA amplification method,1 is indispensable in modern biotechnology and molecular biology.2 PCR is widely used in diagnostics, cloning, biosensors, and many other molecular biology applications.3-4 However, the specificity and efficiency of PCR are often compromised by unintended (re)annealing of singlestranded DNA (ssDNA) (e.g., primer dimerization, false priming, and reannealing of PCR amplicons).5-6 Several nanomaterials have been used to enhance the efficiency and specificity of PCR, including gold nanoparticles,7-9 carbon nanotubes,10 carbon nanopowder,11 graphene nanoflakes,12 cadmium–telluride quantum dots,13 graphene quantum dots,14 dendrimers,15 and titanium dioxide.16 For example, graphene nanoflakes increase PCR yield by enhancing thermal conductivity in the PCR mixture,12 and gold nanoparticles reduce the formation of nonspecific products via DNA and protein adsorption.8 However, these methods have not fully addressed the underlying mechanism for enhanced specificity and efficiency of PCR in the presence of each


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nanomaterial. In addition, gold nanoparticle has been controversial for its role of PCR specificity enhancement.8 Graphene oxide (GO), a material with a honeycomb structure, is prepared by oxidation of a single planar sheet of graphite (i.e., graphene).17 The GO surface contains epoxy, hydroxyl, and carboxyl groups, which impart water solubility.18-20 GO interacts with single-stranded nucleic acids by π stacking and hydrogen bonding but has low affinity for double-stranded nucleic acids.21-22 Owing to these properties, GO has been widely used in applications such as DNA detection,23-24 biosensors based on fluorescence resonance energy transfer,25-26 and real-time monitoring of fluorescent nucleic acids.27-30 During replication of DNA in cells, single-stranded DNA binding proteins (SSBs) inhibit reannealing of single strands of template DNA (Scheme 1a).31 However, artificial DNA amplification methods such as PCR lack SSBs or other components that preferentially bind to ssDNA. Thus, as PCR progresses, reannealing of amplified ssDNA may interfere with further DNA amplification. We hypothesized that GO, a ssDNA-adsorbing material32, would behave analogously to SSBs, thereby enhancing the efficiency and specificity of PCR. However, GO is insoluble in high salt solutions containing Mg2+ such as PCR mixtures33, and it adsorbs protein (e.g., DNA polymerase) through non-covalent interactions.34-36 Divalent cations such as Mg2+ has been known to lead aggregation of the GO due to the strong cross linking of GO sheets by the divalent cation.33 GO could be aggregated by divalent cations such as Mg2+, which is contained in PCR mixture in addition to other salts for buffering. In addition, several studies have reported that GO bound with proteins could cause protein aggregation37, structure distortion38 and loss of function39. Thus, to enhance the solubility of GO in solutions with high


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salt concentration and to minimize nonspecific protein adsorption, we fabricated nano-sized GO (nGO) and coated the surface of nGO with polyethylene glycol (PEG-nGO; Scheme 1b).40 PEG is known as a biocompatible polymer for its resistance to protein adsorption.41-42 Recently, PEG-nGO has been reported to significantly reduce protein adsorption, in which PEGylation on surface of GO can generate a nano-bio-interface for protein interaction.43 Thus, PEG-nGO is expected to modestly interact with protein without inducing conformation damage and dysfunction of protein. We hypothesized that PEG-nGO would adsorb single-stranded primer and template DNA during the denaturation step of PCR (Scheme 1c), thus preventing primerdimer formation during early PCR cycles, when primers are in excess, and inhibiting reannealing of other DNA strands during later cycles, when amplified PCR product has accumulated. Herein, we report that PEG-nGO enhances the specificity and efficiency of PCR under conditions that often generate nonspecific amplicons, such as multiple-round PCR and PCR at low annealing temperatures.

2. EXPERIMENTAL SECTION 2.1. Preparation of PEGylated nano-sized graphene oxide (PEG-nGO). GO (HCGO-W-175) was purchased from Graphene Laboratories, Inc. (Ronkonkoma, NY, USA) and 6-arm polyethylene glycol-amine (15 kDa) was purchased from SunBio (Seoul, Korea). GO (5 mgmL−1) was diluted to a concentration of 2 mgmL−1 and then cracked into nGO by tip sonication for 5 h in an ice bath. For PEGylation, NaOH (1.2 g) and chloroacetic acid (1.0 g) were added to the nGO suspension (5 mL) and bath sonicated for 4.5 h. This step converts –OH groups on the nGO surface to –COOH groups via conjugation of acetic acid moieties, resulting in carboxylated nGO (HOOC-nGO). The HCOO-nGO solution was neutralized by repeated


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rinsing with distilled water and purified by filtration using a 0.2 µm filter membrane (Millipore, USA). The HOOC-nGO solution was diluted with water to an optical density of 0.4 at 808 nm. A solution of 6-arm PEG-amine (2 mgmL−1) was added to the HOOC-nGO solution and the mixture was bath sonicated for 5 min. Next, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride was added to 5 mM. The reaction mixture was stirred for 12 h, and the reaction was terminated by adding mercaptoethanol (50 mM). The mixture was dialyzed in distilled water for 12 h and then centrifuged at 10,000 × g for 1 h in phosphate-buffered saline. The supernatant containing PEG-nGO was saved and stored at 4°C for further use.

2.2. Characterization of GO, nGO, and PEG-nGO. The prepared graphene materials (GO, nGO, and PEG-nGO) were characterized by atomic force microscopy (XE-100 AFM; Park Systems, Seoul, Korea) and Fourier transform infrared spectroscopy (Tensor 27 FT-IR spectrometer; Bruker, Billerica, MA, USA).

2.3. Single-stranded DNA (ssDNA) adsorption and DNA polymerase binding to graphene materials. ssDNA (10 nM, 95-mer) labeled with fluorescein isothiocyanate (FITC) was mixed with increasing concentrations (0, 1, 5, 10, 15, 20, 30, 40, and 50 µgmL−1) of GO, nGO, or PEGnGO and heated at 95°C for 10 min. The reaction mixture was placed in a 96-well plate (SPL Life Sciences Inc., Gyeonggi-do, Korea) and the fluorescence intensity was measured (λex = 485 nm and λem = 535 nm) using a multi-label plate reader (VICTOR X3®; PerkinElmer, Waltham, MA, USA). Binding of Taq DNA polymerase to graphene materials was examined by mixing GO, nGO, or PEG-nGO (0, 5, 10, and 20 µgmL−1) with Taq DNA polymerase (0.2 µgµL−1) and


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heating the mixture at 95°C for 10 min. The mixture was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 8%) with Coomassie Brilliant Blue staining.

2.4. RNA extraction and reverse transcription. Total RNA was extracted from cultured human cancer cells (human leukemia cell line K562; American Type Culture Collection) using TRIzol® Reagent (Invitrogen, Carlsbad, CA, USA). One microgram of RNA was reverse transcribed using a PrimeScript™ 1st strand cDNA Synthesis Kit (TaKaRa Bio Inc., Shiga, Japan). The complementary DNA (cDNA) was stored at −20°C for subsequent use.

2.5. Polymerase chain reaction (PCR). DNA oligonucleotides used in this study were synthesized and purified by Cosmogenetech Inc. (Seoul, Korea). Sequences of primers used for PCR amplification (110 bp of GAPDH cDNA) were as follows: 5′-TTG TTG CCA TCA ATG ACC CCT TCA TTG ACC-3′ (forward primer) and 5′-CTT CCC GTT CTC AGC CTT GAC GGT G-3′ (reverse primer). PCR was performed with 3 µL of cDNA (150 ng) in a total volume of 30 µL containing 0.083 UµL−1 Ex Taq® polymerase (TaKaRa Bio Inc., Shiga, Japan), 0.25 mM deoxynucleotides, 1× Ex Taq® buffer, 100 nM PCR primers, and various concentrations of PEG-nGO. PCR for various size amplicon DNAs was performed with linearized plasmid DNA (pET22b+; Merck Millipore, Darmstadt, Germany) as template and various primer DNAs (shown in Figure 5; sequences in table S1). Linearized plasmid DNA was prepared by digestion with EcoRI (TaKaRa Bio Inc.) at 37°C for 1 h, which was purified through ethanol precipitation. PCR amplification was performed using the following cycling program: pre-denaturation at 95°C for 5 min, followed by 30 cycles of 30 s at 95°C, 30 s at 60°C, and 1 min at 72°C, and a final


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step of 8 min at 72°C. PCR product was separated by agarose gel electrophoresis and stained with ethidium bromide. The intensity of DNA bands was quantified by ImageJ software (http://rsb.info.nih.gov/ij/index.html).

2.6. Melting curve analysis of PCR-amplified DNA. Melting temperatures (Tm) were determined using a real-time thermocycler (Rotor-Gene Q; Qiagen, Hilden, Germany). Each 20 µL sample contained 1× SYBR Green I (Invitrogen, Carlsbad, CA, USA), 5 µL of the 110-bp GAPDH amplicon (0.16 µgµL−1), and PEG-nGO at 0, 1, 3, or 5 µgmL−1. The temperature was increased from 25°C to 99°C and the fluorescence change (λex = 470 nm and λem = 510 nm) was monitored at 0.5°C increments.

3. RESULTS AND DISCUSSION First, we characterized GO, nGO, and PEG-nGO using atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FT-IR). GO particles had a wide size distribution (around 400–1000 nm), whereas nGO and PEG-nGO particles had a narrower distribution and were smaller (about 200 nm) (Figure 1a). AFM demonstrated that the thickness of GO and nGO was about 1.2–1.6 nm, corresponding to approximately a single layer of graphene. In contrast, the thickness of PEG-nGO increased to 4–5 nm owing to PEG conjugation to the nGO surface. Compared with the FT-IR spectra of GO and nGO, the PEG-nGO spectrum showed new amide carbonyl (~1650 cm−1) and methylene (~2800 cm−1) bands, confirming the covalent grafting of PEG chains to the surface of nGO sheets (Figure 1b).


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Next, we investigated whether PCR was compatible with the presence of GO, nGO, or PEGnGO. We performed PCR to amplify glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA, which was obtained from cellular mRNA, in the presence of each graphene material (Figure 1c). No GAPDH amplicon was detected in the presence of GO or nGO, while the presence of PEG-nGO did not hamper cDNA amplification. Interestingly, while nonspecific bands were observed with PEG-nGO at a concentration of 1 µgmL−1, only a single specific band was observed with PEG-nGO at 5 µgmL−1. However, when excess PEG-nGO (10 µgmL−1) was added to the PCR mixture, the reaction was inhibited. We have also confirmed that simple addition of PEG to nGO as a mixture (PEG + nGO) did not assist PCR (Figure S1). These results suggest that PEG-nGO at an optimal concentration is compatible with PCR and enhances specificity, such that only the intended amplicon is produced. To explain the differences in PCR compatibility among the graphene materials, we examined the affinity between PCR components (ssDNA and Taq DNA polymerase) and GO, nGO, and PEG-nGO. PEG-nGO exhibited much weaker affinity for ssDNA than GO and nGO (Figure S2). The apparent Kd values for the interactions between ssDNA and GO, nGO, and PEG-nGO were 0.71, 0.87, and 10.93 µgmL−1, respectively. Taq DNA polymerase was adsorbed onto GO and nGO, whereas PEG-nGO did not adsorb the protein at the concentrations used in the PCR (Figure S3). These data indicate that PEG-nGO has a weaker affinity for ssDNA or DNA polymerase than GO and nGO, making PEG-nGO suitable for PCR. Strong adsorption of ssDNA and Taq DNA polymerase onto GO and nGO surfaces is believed to inhibit DNA amplification. Next, we performed PCR with increasing concentrations of PEG-nGO to determine the optimal concentration for enhancement of PCR specificity (Figure S4). Nonspecific bands diminished as the concentration of PEG-nGO increased from 0 to 5 µgmL−1. When excess PEG-nGO (10


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µgmL−1) was added to the PCR mixture, no amplified DNA bands were observed. The GAPDH band was most prominent at 5 µgmL−1 PEG-nGO. Thus, 5 µgmL−1 was chosen as the optimal PEG-nGO concentration for subsequent experiments. About 30% of ssDNA was adsorbed at 5 µgmL−1 PEG-nGO (Figure S2), suggesting that PEG-nGO may adsorb primers and singlestranded template DNA during PCR, thereby facilitating PCR by reducing primer dimerization and reannealing of amplified DNA strands. Consecutive rounds of PCR often produce a multitude of nonspecific amplicons, which appear as smeared DNA bands in agarose gels. This is caused by a high concentration of amplified template from the previous PCR round and use of the same primers for the second-round PCR. To address this problem, inner primer sets (nested primers) are generally used for second-round PCR.44 To test the effect of PEG-nGO on the efficiency and specificity of consecutive rounds of PCR, we performed second-round PCR with or without PEG-nGO, using serial dilutions of the first-round PCR product and the same primers used in the first round of PCR (Figure 2a). When second-round PCR was performed in the presence of PEG-nGO, discrete target DNA bands, without smearing, were observed at all dilutions of the first-round PCR product. In the absence of PEG-nGO, however, smeared bands corresponding to nonspecific PCR products were observed at all dilutions. These results suggest that PEG-nGO enhances the specificity and efficiency of second-round PCR performed with first-round primers, thus eliminating the need for nested PCR. To demonstrate that PEG-nGO indeed enhances the efficiency of successive rounds of PCR with the same PCR primers, we carried out further rounds of PCR in the presence or absence of PEGnGO (Figure 2b). In this experimental setup, the previous-round PCR product was used as DNA template without dilution. Nonspecific DNA amplification (smearing) was observed in all


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reactions without PEG-nGO, whereas the presence of PEG-nGO reduced nonspecific DNA amplification, yielding a single DNA band at multiple rounds of PCR (from the 2nd round to the 4th round PCR). However, loss of PCR specificity was observed at the 5th round of PCR. We postulated that additional rounds of PCR needs more PEG-nGO, in which higher concentration of PEG-nGO is needed to enhance PCR specificity when template DNAs are initially present at excess amounts (i.e. multiple-rounds PCR amplicon). When the 5th round PCR was performed with increasing concentrations of PEG-nGO (0, 5, 10, 15 µgmL−1), higher concentration of PEGnGO (15 µg/mL) was needed for specific PCR amplification (Figure S5). Thus, repeated rounds of PCR with excess amount of heat-denatured ssDNA templates would require a higher concentration of PEG-nGO to support specific amplification of target DNA template. Next, to address whether PEG-nGO concentration needs to be changed depending on primer concentration, PCR was performed in the presence or absence of PEG-nGO (5 µgmL-1) with primer DNA at various concentrations (Figure S6). Nonspecific PCR amplification bands were observed at all reactions in the absence of PEG-nGO, except for 0.05 µM of primer DNA (the lowest primer concentration). However, presence of PEG-nGO significantly reduced nonspecific DNA amplification which was observed in PCR with higher primer DNA concentrations (>0.05 µM). This result indicates that the optimal concentration of PEG-nGO (5 µgmL-1) chosen for a conventional PCR can enhance specificity of DNA amplification in a wide range of primer DNA concentrations, albeit PEG-nGO is not needed for PCR with very low primer DNA concentration. In conventional PCR, nonspecific products can be generated by primer interactions, notably primer dimerization. Primer-dimers can be produced when the primer concentration is high or when the forward and reverse primers have 3′ complementarity.5, 45 Since PEG-nGO adsorbed ssDNA in a dose-dependent manner (Figure S2), we hypothesized that PEG-nGO might adsorb


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excess primers, thus mitigating primer dimerization. To test our hypothesis, we added primers for GAPDH cDNA amplification to a PCR mixture without template DNA and carried out temperature cycling. As shown in Figure 3a, amplified DNA was generated from dimerized primers in the absence of target DNA template (i.e., GAPDH cDNA). However, primer-dimer formation and amplification decreased significantly with increasing PEG-nGO concentration. Dimerized primer bands completely disappeared in the presence of 5 µgmL−1 PEG-nGO (the optimal concentration in our PCR system). Thus, PEG-nGO adsorbs excess primer DNAs in the first step of conventional PCR and prevents primer dimerization during temperature cycling. We next examined the effect of PEG-nGO on the specificity of PCR at low annealing temperatures (Figure 3b). Primers can anneal to template DNA strands more easily at lower temperatures, which increases the yield of PCR products but decreases the specificity of DNA amplification. We observed many nonspecific DNA bands (smearing) in the absence of PEGnGO at low annealing temperatures. However, these nonspecific bands were not observed in the presence of PEG-nGO. This result indicates that PEG-nGO decreases mispairing between primers and template DNA, likely by adsorbing excess ssDNA in the PCR mixture and thus maintaining appropriate ssDNA concentrations during temperature cycling. The fidelity of DNA replication is an important factor in DNA amplification. During DNA replication in cells, multiple SSBs bind to ssDNA after double-stranded DNA (dsDNA) is unwound by DNA helicase, ensuring high fidelity and efficiency of DNA replication by inhibiting reannealing of ssDNA.31 Inspired by the similar properties of SSBs and PEG-nGO, we hypothesized that PEG-nGO, like SSBs, could improve PCR specificity and efficiency by inhibiting reannealing of amplified DNA during the annealing step and facilitating DNA melting during the denaturing step. To test this hypothesis, we examined the effect of PEG-nGO on the


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reannealing of PCR-amplified DNA by performing second-round PCR with or without primers (Figure 4a). Nonspecifically amplified DNA with high molecular weight was observed in reactions without primers, indicating that excess template DNA could anneal nonspecifically and generate nontarget products. These nonspecific DNA bands diminished with increasing PEGnGO concentration and completely disappeared in the presence of 10 µgmL−1 PEG-nGO. However, a specifically amplified target DNA band was observed in the agarose gel when second-round PCR was performed in the presence of primers and 10 µgmL−1 PEG-nGO. This result suggests that PEG-nGO inhibited nonspecific reannealing of amplified DNA during PCR. We next examined the effect of PEG-nGO on the melting temperature (Tm) of PCR products by measuring the amount of dsDNA as a function of temperature (Figure 4b). GAPDH cDNA was amplified by PCR in the presence of various amounts of PEG-nGO. The temperature of the PCR mixture was raised from 25°C to 95°C and the relative amount of double-stranded amplified DNA was quantified with SYBR Green I. Derivative melting curves of double-stranded amplified DNA are shown in Figure 4b. Tm values were negatively correlated with the concentration of PEG-nGO, indicating that PEG-nGO can facilitate dissociation of dsDNA. We also investigated whether PEG-nGO could speed up the dissociation of PCR-amplified dsDNA (Figure 4c). Temperature cycling was reduced to two steps with a shortened denaturation time (30 cycles of 1 s at 95°C and 15 s at 60°C). Control PCR (30 cycles of 30 s at 95°C, 30 s at 60°C, and 1 min at 72°C) was also performed (lanes C in Figure 4c). In the presence of PEGnGO, an amplified DNA band was clearly visible, without nonspecific DNA amplification, after the first and second rounds of PCR, whereas both rounds of PCR performed without PEG-nGO produced smeared DNA bands. These data indicate that PEG-nGO can make thermal cycling quicker and simpler by facilitating the dissociation of double-stranded PCR products.


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To address whether PCR specificity enhancement with PEG-nGO can be substantiated for PCR for various size of target DNA amplification, we conducted the second-round PCR with or without PEG-nGO using the first-round PCR products of various target size (102, 198, 401, 805, 1600 bp) as template DNAs (Figure 5). Using a plasmid DNA as template for PCR of various amplicon sizes, the first PCR amplicon products were clearly visible without nonspecific amplification (Figure 5b), whereas successive round of PCR (the 2nd round PCR) produced nonspecific DNA amplification with smearing DNA bands (Figure 5c). In the presence of PEGnGO (5 µgmL−1), however, PCR of various amplicon sizes were accomplished without any nonspecific DNA amplification, in which amplicon DNA bands was clearly visible without smearing (Figure 5d). These results demonstrate that PEG-nGO of optimal concentration increases specificity and efficiency in PCR regardless of target amplicon size. Taken together, the results presented here support our hypothesis that PEG-nGO interrupts reannealing of double-stranded template strands by preferentially binding to denatured ssDNA, which is analogous to the function of SSBs. Our hypothesis that ssDNA-favoring PEG-nGO increases PCR efficiency was further supported by earlier observation that SSB can greatly enhance PCR efficiency.46

4. CONCLUSION In summary, we examined the effects of PEG-nGO on the specificity and efficiency of PCR. The results of this study indicated that PEG-nGO, at an optimal concentration, significantly improves the specificity and efficiency of PCR for target DNA amplification in diverse sizes. The mechanism underlying the facilitation of PCR by PEG-nGO was elucidated by investigating interactions between PEG-nGO and ssDNA. First, in the initial stage of PCR, PEG-nGO


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decreases primer dimerization and nonspecific annealing between primers and template DNA by adsorbing excess primers. Second, in the later stage of PCR, when amplified PCR products have accumulated, PEG-nGO attenuates reannealing of double-stranded amplified DNA by preferentially binding to denatured ssDNA, thus facilitating annealing of primers to template strands. Third, owing to its preferential interaction with ssDNA, PEG-nGO can shorten the time required for denaturation by facilitating melting of double-stranded amplified DNA, which serves as template for subsequent rounds of PCR. Hence, PEG-nGO enhances the specificity and efficiency of PCR by preferentially binding to ssDNA, which is analogous to the role of SSBs in DNA replication in vivo.


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Scheme 1. Schematic of enhancement of PCR specificity by PEG-nGO. (a) Role of SSBs in DNA replication as inhibitors of strand reannealing. (b) Expected structure of PEG-nGO. (c) Effect of PEG-nGO on PCR specificity; PEG-nGO binds preferentially to ssDNA during temperature cycling in PCR and decreases nonspecific annealing.


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Figure 1. Polyethylene glycol-engrafted nano-sized graphene oxide (PEG-nGO). (a) AFM images of GO, nGO, and PEG-nGO deposited on mica substrate. Image of nGO was taken before filtration (200 nm pore size filter), and image of PEG-nGO showed relatively even size distribution of ~150 nm. (b) FT-IR spectra of GO, nGO, and PEG-nGO. (C) Effect of GO, nGO, and PEG-nGO on PCR. PCR was performed in the presence of 1, 5, or 10 µgmL−1 GO, nGO, or PEG-nGO to amplify human GAPDH cDNA (110 bp). PCR products were analyzed by agarose gel electrophoresis (2%) with ethidium bromide staining. Numbers in the gel image indicate the relative band intensity of the GAPDH amplicon. M, DNA markers.


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Figure 2. Effect of PEG-nGO on the specificity and efficiency of consecutive rounds of PCR. (a) Effect of PEG-nGO on the specificity of second-round PCR. First-round PCR was performed without PEG-nGO, and the amplified product (GAPDH cDNA, 110 bp) was serially diluted and used as template DNA for second-round PCR in the presence or absence of PEG-nGO (5 µgmL−1). C, control DNA (first-round PCR product); P, primer DNA; M, DNA markers. PCR products were analyzed by agarose gel electrophoresis (2%) with ethidium bromide staining. (b) Effect of PEG-nGO on the efficiency of multiple-round PCR. First-round PCR was performed without PEG-nGO, and the PCR product (GAPDH amplicon) was used as template for the next round of PCR in the presence or absence of PEG-nGO (5 µgmL−1).


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Figure 3. Effect of PEG-nGO on primer dimerization and false priming in PCR. (a) Prevention of primer dimerization with PEG-nGO. PCR without template DNA was performed with two different primer DNA concentrations (0.1 and 1 µM) in the presence or absence of PEG-nGO (0, 1, 3, 5, 10, or 20 µgmL−1). Amplified DNA was analyzed by polyacrylamide gel electrophoresis (10%) with ethidium bromide staining. DNA bands appearing above the primer DNA represent nonspecifically amplified DNA resulting from primer dimerization. (b) Effect of different annealing temperatures (30, 40, and 50°C) on the specificity of PCR amplification of 110 bp of human GAPDH cDNA with or without PEG-nGO (5 µgmL−1). PCR products were analyzed by agarose gel electrophoresis (2%) with ethidium bromide staining. M, DNA markers.


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Figure 4. PEG-nGO inhibits reannealing of heat-denatured template DNA and facilitates dissociation of PCR product. (a) Effect of PEG-nGO on the specificity of second-round PCR. First-round PCR was performed without PEG-nGO, and the DNA amplicon (GAPDH cDNA) was used as template for second-round PCR in the presence or absence of PEG-nGO with or without primers (1.0 µM). Lane C, control DNA (110-bp GAPDH amplicon); P, primer DNA; – Taq, PCR without DNA polymerase. (b) PEG-nGO facilitates DNA melting. Fluorescence was measured during thermal denaturation of the PCR product (110-bp GAPDH amplicon) using real-time PCR equipment, and melting temperatures (Tm) were determined from plots of the negative first derivative of fluorescence versus temperature (−dF/dT vs. T). Samples for thermal denaturation contained 1× SYBR Green fluorescent dye, GAPDH amplicon (0.16 µgµL−1), and PEG-nGO at 0, 1, 3, or 5 µgmL−1. The temperature was increased from 25°C to 99°C and the fluorescence change (λex = 470 nm and λem = 510 nm) was monitored at 0.5°C increments. (c) Effect of shortened denaturation time during temperature cycling on the specificity of first- and second-round PCR. Temperature cycling consisted of 30 cycles of 1 s at 95°C and 15 s at 60°C. Control PCR without PEG-nGO was performed with a cycling program of 30 cycles of 30 s at 95°C, 30 s at 60°C, and 1 min at 72°C (lanes C). M, DNA markers.


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Figure 5. Effect of the PEG-nGO on PCR at various size of target amplicon (a) Schematic diagram of various sized PCR products. (b) PCR was performed using plasmid DNA as template (pET22b+, 0.75 ng) to amplify various size target (102, 198, 401, 805, 1600 bp) in the absence of PEG-nGO. M, DNA markers. (c) Second round PCR was performed without PEG-nGO to amplify various amplicon DNAs. Each DNA amplicon obtained in the 1st round PCR was used as template for the second round PCR. (d) Second round PCR was performed in the presence of PEG-nGO (5 µgmL−1). \


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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: Oligonucleotide primers in PCR, comparison of PEG-nGO and a simple mixture of PEG and nGO for enhancing PCR specificity, comparison of binding affinities between ssDNA and graphene materials (GO, nGO, and PEG-nGO), binding of Taq DNA polymerase to graphene materials (GO, nGO, and PEG-nGO), optimization of PEG-nGO concentration for enhancement of PCR specificity, optimization of PEG-nGO concentration for enhancement of 5th-round PCR specificity, effect of PEG-nGO on PCR at different concentrations of primer DNA (PDF) AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by a National Research Foundation grant funded by the Korean Government (NRF-2014R1A2A1A-11051361).


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Facilitation of Polymerase Chain Reaction with Poly(ethylene glycol

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