Size analysis of large DNA molecules by relaxation time measurement using a nanoslit channel

Determining the size of DNA molecules >10 kbp continues to be challenging, as conventional methods, such as pulsed-field gel electrophoresis require time-consuming analysis and artificial gel structures increase the risk of mechanical fragmentation to DNA molecules during repeated hooking and stretching. Herein, we developed a new analytical method for identifying the size of DNA molecules by measuring the time required for a stretched large DNA molecule to relax into a random coil, using nanoslit channels with depths of 130-49 nm. By maintaining the initial stretching ratio of the collected DNA molecules at <30%, we successfully differentiated a mixed DNA sample containing λ and T4 DNAs into two distinct peaks in the relaxation-time histogram. Furthermore, we explored the influence of the number of collected relaxation times and nanoslit depths on the resolution of size analysis. Our findings indicated that the nanoslit depth was the primary factor affecting the size determination resolution. Reducing the nanoslit depth enhanced resolution, whereas the number of effective relaxation times did not impact the resolution once a critical data threshold was reached. A nanoslit channel of depth 49 nm exhibited superior performance, with a maximum resolution of 2.33 and a short analysis time of 60 s, surpassing both conventional methods in terms of resolution and time efficiency. The proposed method shows great potential for accurate, large-scale DNA size analysis.