Title : Manipulation and analysis of large DNA molecules using micro/nanogap structures
Abstract:
Analyzing the length of DNA molecules is one of the fundamental processes for analyzing DNA molecules of bacteria and viruses in the human bodies and in the environment. For example, to identify the genotype of bacteria, analyzing the length distribution of DNA fragments is required after its genomic DNA is cut at the positions of specific base sequences using the restriction enzymes. To analyze the horizontal transmission of bacterial genes, analyzing the length distribution of multiple plasmid DNAs in the bacteria is required as a pretreatment for gene analysis. These analyses have been used to identify the infection routes and the gene propagation pathways of dangerous bacteria such as drug-resistant bacteria, and require high-speed and high-sensitivity analysis. Gel electrophoresis, which uses the mesh structure of the gel as a molecular sieve matrix, has been usually used to analyze the length of DNA molecules. However, this method requires a large consumption of DNA samples and long analysis time. In particular, the length analysis of large DNA, which is larger than 10 kbp (base pairs), such as bacterial genomic DNA and plasmid DNA has required several days to be analyzed. This is because large DNA molecules have a random coil shape with a diameter of several micrometers and are greatly deformed when they migrate in the small mesh structure of a gel, requiring slow migration using a special electrophoresis method. The unique deformation of large DNA molecules is a difficulty in high-speed analysis. Therefore, we have developed the methods to analyze large DNA molecules by manipulating them using micro- and nano-scale gaps formed in a microfluidic channel. We have proposed the methods to control the Brownian motion of large DNA molecule using an array of microgap structures, to trap large DNA molecules using a nanoslit structure with the depth of several tens of nanometers, and to stretch a single large DNA molecule. By manipulating large DNA molecules using these micro- and nano-gaps, fast length analysis of large DNA molecules at the tens of minutes level has been realized. In this presentation, we would like to introduce the principles, the experimental results, and impact of our micro/nanogap -based DNA analysis methods. If these methods can be put into practical use, we expect that there are various applications, not only for bacteria and viruses, but also for extracellular vesicles and environmental DNA, and so on.
Audience Take Away:
- Micro/Nanostructures fabrication using a micro-fabrication technique
- Migration Control of biomolecules using micro/nanostructures
- Understanding of methods of analysis of large DNA molecules
