Aqueous suspension of carbon nanotubes enhances the specificity of long PCR. Zhizhou Zhang, Cencao Shen, Mingchun Wang, Han Han, and Xiaohong Cao.
Research Reports
Aqueous suspension of carbon nanotubes enhances the specificity of long PCR Zhizhou Zhang, Cencao Shen, Mingchun Wang, Han Han, and Xiaohong Cao BioTechniques 44:537-545 (April 2008) doi 10.2144/000112692
DNA manipulation technology is facing more challenges in the postgenomics era. More and more nanomaterials have been investigated for their potential implications in developing better gene technology. In this study, we reported the beneficial effect of single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) in enhancing the specificity and total efficiency of long (14 kb) PCR. Hydroxylic and carboxylic carbon nanotubes (CNTs) had similar enhancing effects. Nanotubes could become another component for improvements in the amplification of long DNA.
INTRODUCTION A PCR target longer than 5–6 kb can usually be called long PCR. Long PCR has always been a problem for many laboratories of biomedical sciences worldwide. Although there are some commercially available long PCR kits, those kits often do not work for the amplification of specific long DNA fragments. There have also been some reports that describe protocols to improve the performance of long PCR (1–3), but in most cases, those protocols are very complex and not convenient to follow. Long PCR technology therefore has much room to improve. In practice, different approaches have to be tried before a long DNA fragment is finally able to be well amplified. There are already a number of PCR enhancers, such as dimethyl sulfoxide, glycerol, formamide, betaine, singlestranded DNA binding proteins, and nanogold particles (4,5). However, how to correctly use those enhancers for long PCR remains unclear. Recently, it has been suggested that single-walled carbon nanotubes (SWCNTs) can act similarly to Mg2++ ions in maintaining the high activity of DNA polymerase in PCR (6), but the potential effects of different types of carbon nanotubes (CNTs) on long PCR remain to be elucidated. Long PCR has become increasingly important in the postgenome era,
partially because genome technology, rather than simply gene technology, is experiencing some great technological challenges. No mature technology has been developed to easily amplify very long, say 100 kb, DNA sequences, let alone GC-rich long DNA fragments. Long DNA manipulation technologies, including long PCR, are critical for cloning and other molecular manipulations of very long genomic sequences in functional genomics studies. In this report, the effect of carbon CNTs on a long PCR system is explored. We amplified a 14-kb fragment from a λ DNA template and found that CNTs significantly improved the amplification efficiency. MATERIALS AND METHODS Carbon Nanotubes All CNTs [SWCNTs, multi-walled carbon nanotubes (MWCNTs), hydroxylic CNTs, or carboxylic CNTs (CNT-OH, CNT-COOH, respectively)] were purchased from TimesNano (Chengdu, China). The average outside diameters and product numbers are presented in Table 1. Sterilized distilled water was used to prepare all CNT suspensions with the final concentration of 10 mg/mL (w/v). All CNT suspen-
sions need to be ultrasonicated at 100 W for 1–2 h just before adding to the PCRs. PCR System In the long PCR system, primer P3 5′-TGGTTTATTGGAGTAGATGCTTG-3′′ and primer P4 5′-GAGAGTTGTTCCGTTGTGGG-3′ were designed to amplify a 14.3-kb fragment from λ DNA. PCR reagents were mixed in a final volume of 25 μL in 200-μL thin-walled tubes according to the following conditions: 1× × PCR buffer (10 mM Tris, pH 8.8 at 25°C, 50 mM KCl, 0.08% Nonidet P40, 2.8 mM MgCl2), 0.5 mM dNTP, 0.35 μM μ M P3 and P4, 2 ng/ ng/μ μL λ DNA, 0.1 U/ μL U/μ μL μL Taq DNA polymerase, 0.05 U/μL Pfu DNA polymerase, and the final volume was filled with aqueous suspensions of the CNTs and distilled water. PCR was performed on a T-Gradient Thermal block (Biometre, Göttingen, Germany) and/or a SpeedCycler (Analytik Jena AG, Jena, Germany). Initial denaturation at 92°C for 2 min was followed by 32 cycles of denaturation at 92°C for 20 s, annealing at 63°C for 50 s, and extension at 72°C for 15 min. The final step was an extension at 72°C for 10 min. Trehalose (T5251) and betaine (B2629) were purchased from Sigma (St. Louis, MO, USA). λ DNA, dNTP, Taq DNA polymerase, and Pfu DNA polymerase were from BBI (Markham, ON, Canada). Primers were synthesized
Teda Bio-X Center for Systems BioTechnology, Tianjin University of Science and Technology, Tianjin, China Vol. 44 ı No. 4 ı 2008
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Research Reports from Sunbiotech (Beijing, China). All other DNA polymerases such as KOD Dash, DeepVent, and DeepVent (exo-) were purchased from Sangon (Shanghai, China). PCR Efficiency Assessment Three μL of PCR product were examined with 0.7% (w/w) agarose gel with standard protocols. At the end of the electrophoresis, the gels were stained by ethidium bromide (Amresco, Solon, OH, USA) with the concentration of 5 μg/mL in a 1× × Tris-acetate-EDTA (TAE) solution (5). PCR product was photographed and quantitatively scanned by Gel Doc EQ (Bio-Rad Laboratories, Hercules, CA, USA). In all figures, molecular weight markers (M) represented the same λ DNA Marker (λDNA/ λDNA/Hin λDNA/ DNA/HindIII) (Sangon, Shanghai, China). Preliminary Fidelity Assessment with DNA Sequencing A series of nanoparticles including MWCNTs and SWCNTs were used as PCR additives. PCR product was gelpurified followed by phenol/chloroform extraction and ethanol precipitation according to standard protocols. Purified DNA samples were sequenced with Sanger’s method at Sunbiotech. RESULTS CNTs Significantly Improve Long PCR Efficiency In this long PCR model system, we usually obtain nonspecific products, as manifested by smear bands in electrophoresis, even after adding a variety of PCR additives (data not shown). In order to get appropriate conditions of the Mg2+ concentration and the annealing temperature, betaine, a classical additive for long PCR, was applied in our model system as a starting condition. Annealing temperature gradients of 55°–70°C with a fixed Mg2++ concentration of 2.8 mM, or various concentrations of Mg2++ from 2.3 to 5.3 mM with a fixed annealing temperature of 61°C, were first tested as shown in Figure 1A. Based on these results, 61°C and 2.8 mM Mg2++ were chosen as the optimal 538 ı BioTechniques ı www.biotechniques.com
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Figure 1. Carbon nanotubes (CNTs) significantly improved long PCR efficiency. Expected PCR bands are marked by an arrow. (A) Effects of Mg2+ concentration and annealing temperature in the presence of 1.2 M betaine without CNTs. (B) Effect of 1–2 nm single-walled carbon nanotubes (SWCNTs) on the specificity of PCR. (C) Effect of
