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Researchers Perform PCR on DNA Bound to Single-walled Carbon Nanotubes

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Researchers at West Virginia University have begun to elucidate the parameters for PCR of DNA interacting with single-walled carbon nanotubes.

In exploratory research published in PLoS ONE this month, the scientists found that DNA directly associated with SWCNTs was amplifiable by PCR at some concentrations. The work has potential applications for PCR that exploits the SWCNT-DNA association, as well as for understanding how SWCNT contamination in the environment or in a diagnostic device might inhibit a PCR reaction.

Lead author of the study, Letha Sooter, a member of Nanotechnology Sensing Advances in Field and Environment (NanoSAFE), WVU's initiative for nanoscale science and engineering, said part of her rationale for the study was to see how nanotubes may interfere with PCR.

The study mixed SWCNTs with DNA coding for an estrodiol molecular recognition element at different concentrations. It then used primers for the estradiol recognition element and Taq polymerase-based amplification to detect the DNA mixed with SWCNTs. It examined how the PCR reaction was effected by different SWCNT configurations, levels of SWCNT dispersion, the DNA sequence used for dispersion, and SWCNT concentration, as well as whether or not the template DNA was primarily associated with the SWCNT or free in solution, Sooter said in email to PCR Insider.

"With the increasing use of nanotubes in commercial applications, they will increasingly be found in the environment," Sooter said. "Identification and analysis of biological samples often involves the use of PCR. It is important to understand how the presence of SWCNTs, present in the environment the sample was collected from, or present because an analytical device is constructed of them, can influence the success of the PCR reaction."

However, "it may also be that under certain circumstances SWCNTs benefit PCR reactions," she said.

SWCNTs are fullerenes consisting of single atom-thick tubes of carbon, somewhat similar in structure to graphite. They can be millions of times longer than they are wide, and manufactured with different wrapping chiralities. For unknown reasons, they show a predilection for nucleic acids. Long and thin SWCNTs are highly hydrophobic, interacting via van der Waals forces. Long, thin, and charged DNA turns out to be one of the best molecules to disperse SWCNTs so they can go into solution.

As Sooter described it, "Imagine a sheet of paper that is one carbon atom thick. You can roll the sheet of paper into a cylinder, a carbon nanotube, in many different ways. Sometimes the edges of the sheet of paper will line up perfectly, other times the edges may be offset from each other. The different ways you roll the paper are like the different carbon nanotube chiralities. Carbon nanotubes are very lightweight, but they are very strong. They have metallic or semiconductor properties, they can also have unique optical signatures ... [and] each chirality has slightly different properties."

In fact, another study, published recently in the American Chemical Society journal Langmuir, showed binding between single-stranded DNA and carbon nanotubes is strongly dependent upon DNA sequence and nanotube chirality.

Beyond simply using DNA as a dispersal reagent, or measuring SWCNT contamination in the environment, exploiting the natural interaction between DNA and SWCNTs is a growing field.

Recent studies, such as one in ACS Nano by Stuart Lindsay's group at Arizona State University, used optical and electrical detection to show fluorescent molecules moving through SWCNT channels. A study published last December in Nature Communications by researchers at the Chinese Academy of Sciences went one step further, and shot SWCNTs into a lipid bilayer. This study showed SWCNTs manufactured to be "ultra short" have surprising properties, different from their longer brethren. The CAS researchers used a micro-injection probe to insert nanotubes into the bilayer. Much like an ion channel in a neuron, they found that the SWCNTs could pass current across the membrane, as measured with a patch-clamp amplifier.

More importantly, when SWCNTs were made into nanopores, they also passed DNA. The researchers used them to discriminate single strands of DNA containing 5-hydroxymethylcytosine from those with 5-methylcytosine, an otherwise challenging task for conventional bisulfite sequencing that is increasingly important to epigenetic analyses. This may have implications for high-throughput screening of genomic DNA fragments that contain 5hmC, the authors said.

In an email to PCR Insider, lead author on the Nature Communications study, Haichen Wu, emphasized the many potential applications utilizing both the nanopore properties of SWCNTS as well as their natural affiliation with DNA.

"Any DNA-nanopore applications should be possible with SWCNTs ... DNA sequencing, DNA damage sensing, determination of DNA secondary structures, and so on," he said, adding that, "SWCNTs also bind with DNA molecules and thus affect the functions of DNA. One example is the DNA-SWCNT sensor. Also, SWCNTs might be used to modulate DNA-protein interactions and other DNA-related biological processes."

The SWCNT-DNA interaction may ultimately prove useful to nucleic acid analysis methods such as PCR. An aqueous suspension of nanotubes was reported in 2008 to enhance specificity of PCR longer than five kilobases, for example. Another study that same year also demonstrated SWCNTs could improve PCR results.

Virus researcher Ian Lipkin of Columbia University recently told GenomeWeb Daily News his group will be using nanotubes for virus detection. "We are using a fieldable microarray that will not require amplification, either isothermal or PCR-based, but will detect binding events in fluid samples, extracts, and tissues," Lipkin said of one project. He said this effort will incorporate carbon nanotubes to recognize genetic material from oligonucleotides, and is based on probe libraries.

However, future work on SWCNTs must also determine whether their DNA interactions make them genotoxic. A recent paper in Aquatic Toxicology, for example, showed carboxylated SWCNTs preferentially affected some strains of ocean bacteria, but not others.

This means determining the parameters for PCR of SWCNTs bound to DNA is important. Due to their increasing commercial use, this "may mean that an environmental sample you collected contains SWCNTs as a contaminant, or it may mean that some of the materials you are using to prepare or run your PCR reaction contain SWCNT," Sooter said. "We were interested in the effects this might have on a PCR reaction. It may be that at low levels, SWCNT can improve the efficiency of a PCR reaction, while at high levels they inhibit the reaction."

At the moment, PCR of SWCNT-associated DNA is still exploratory. Sooter said this is part of the charm of SWCNT-DNA research. "I enjoyed this study for many reasons, one of which is that it raises so many interesting questions."

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