The ability to detect single molecules of DNA or RNA has gained the attention of scientists as it offers a rich area of exploration of the single most crucial hereditary material. Recently, experts applied nanopore technology for single-molecule DNA detection, providing them with real-time analysis of DNA and RNA strands.
Since this technique is flexible and costs less, it can be used in clinical and research settings whenever samples need to be studied quickly and efficiently. However, this novel technology faces some challenges, such as sample contamination.
Enhanced DNA Filtering System
Improving and optimizing single-molecule DNA extraction using nanopores has been the main focus of researchers from the Tokyo University of Agriculture and Technology. Professor Ryuji Kawano led the team to create a DNA-filtering system utilizing a type of nanopore developed from the alpha-hemolysin (αHL) protein.
The DNA filtering system includes a microdevice to prepare two water-in-oil droplets. The sample droplet containing the target DNAs and the controlling droplet without the DNAs are separated by a lipid bilayer which permits the single-molecule DNA to travel through an αHL nanopore.
According to Kawano, they aim to understand better the problems in achieving accurate counts of single molecules. They believe using αHL nanopores for DNA filtering systems can potentially revolutionize DNA analysis. This is because real-time detection and evaluation of DNA at the single-molecule level are enabled, preventing the need for labeled or partitioned sample solutions.
The researchers proposed several avenues to address the problem of contamination. Since DNA samples can get contaminated from impurities in the air, the researchers tried to implement changes where the work areas were decontaminated with UV light for 15 minutes.
On the other hand, DNA contamination can also occur in the oil and lipid mixture, although the solution to this problem is less straightforward. The research team tried various phospholipids and another technique called the contact bubble bilayer method. Finally, they tested the PCR clamp method, where peptide nucleic acids (PNA) were added to bind to the DNA to create PNA-DNA duplexes. It was found that adding PNAs to the solution reduces DNA contamination by 99.98%.
Nanopore Technology in DNA Sequencing
Nanopore DNA sequencing is a laboratory technique used to determine a DNA molecule's exact sequence of nucleotides. This approach provides a cheaper, quick process for studying long strands of DNA.
The sequence of DNA bases - adenine (A), cytosine (C), guanine (G), and thymine (T) - encodes the biological information used by the cells to develop and operate. In nanopore DNA sequencing, the code of single DNA strands is read while threading through extremely tiny pores embedded within a membrane. As the DNA strands move through the nanopore, signals are created, which will be converted to read each DNA base.
Traditional DNA sequencing methods can only study short strands of DNA which need to be reassembled after the analysis. Nanopore sequencing technology is the only DNA sequencing approach that provides real-time analysis in fully scalable formats that will suit most experimental needs.
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