Single-molecule DNA extraction using nanopore technology offers real-time DNA and RNA analysis. It is a low-cost technique that can be used in clinical and research settings when samples need to be analyzed quickly and efficiently. However, single-molecule DNA extraction may have sample contamination issues; research to minimize sample contamination and optimize the technology with nanopores is ongoing. A recently published paper outlines how a DNA-filtering system using a nanopore can be developed from α-hemolysin protein.
“We set out to develop a DNA filtering system using α-hemolysin (αHL) nanopores and to better understand the challenges faced in achieving accurate single-molecule counting,” said Ryuji Kawano, PhD, a professor at Tokyo University of Agriculture and Technology in Tokyo, Japan. “The development of a DNA filtering system using αHL nanopores has the potential to revolutionize DNA analysis by enabling real-time detection and analysis of DNA at the single-molecule level, eliminating the need for labeling or partitioning sample solutions.”
Overcoming sample contamination issues
In this technique, a microdevice is used to prepare two water-in-oil droplets. One is the sample droplet containing target DNAs and one is the control droplet without the DNAs. The droplets are separated by a lipid bilayer, which allows a single molecule of DNA to move through an αHL nanopore. As the molecules move through this nanopore, they are converted to electrical signals. “We aimed to optimize the experimental environment, reduce the volume of solution containing the target molecule, and use the PCR clamp method to tackle the issue of contamination, which we found to be an almost unsolvable problem in single-molecule counting,” said Kawano.
To solve the problem of contamination, researchers proposed several different avenues. Contamination in DNA samples can come from the air and from impurities in the samples. Improving air-borne contamination proved to be relatively straightforward. The sample DNA was used in a designated experimental area, special pipettes that limited airborne contamination were used, and work areas were decontaminated with UV light for 15 minutes. DNA contamination from the air was completely eliminated after implementing these changes.
The PCR clamp method
DNA contamination also happens within the oil and lipid mixture—solving this problem was challenging. Researchers tried different phospholipids, but this did not have an impact on contamination. Another technique called the contact bubble bilayer method, in which water bubbles are introduced to the oil layer using glass pipettes, was tried, but contamination was detected. This is likely due to DNA molecules being accidentally introduced to the oil and lipid mixture when the emulsion is formed.
Finally, researchers tested the PCR clamp method, which adds peptide nucleic acids (PNA) to bind to the DNA, creating PNA–DNA duplexes. As the duplex moves through the nanopore, they unzip and only the target DNA moves to the next droplet.
The same method is applied to contaminant DNA. PNA is added to the solution so it can bind to the contaminants, but this PNA–DNA duplex doesn’t get unzipped. This makes it easy to tell the target DNA from the contaminant DNA, allowing the results to exclude and disregard the extra DNA. When PNAs were added to the solution, 99.98 percent of DNA contamination got leached away.
Looking ahead, researchers hope to continue optimizing and refining the DNA filtering system. “We aim to overcome the challenges associated with accurate single-molecule counting, particularly the issue of contamination. Our ultimate goal is to develop a reliable and efficient single-molecule filter,” said Kawano.
- This press release was originally published on the Tokyo University of Agriculture and Technology website