Separation of Long DNA Molecules by Quartz Nanopillar Chips under a Direct Current Electric Field

摘要

We have established the nanofabrication technique for constructing nanopillars with high aspect ratio (100-500 nm diameter and 500-5000 nm tall) inside a microchannel on a quartz chip.The size of pillars and the spacing between pillars are designed as a DNA sieving matrix for optimal analysis of large DNA fragments over a few kilobase pairs (kbp).A chip with nanopillar channel and simple cross injector was developed based on the optimal design and applied to the separation of DNA fragmennts (1-38 kbp) and large DNA fragments (gamma DNA,48.5 kbp;T4 DNA,165.6kbp) that are difficult to separate on conventional gel electrophoresis and capillary electrophoresis without a pulsed=field technique.DNA fragments ranging from 1 to 38 kbp were separated as clear bands and furthermore,the mixture of gamma DNA and T4 DNA was successfully separated by a 380-mum-long nanopillar channel within only 10 s even under a direct current (dc) electric field.Theoretical plate number N of the channel (380-1450 mum long) was 1000-3000 (0.7X10~6-2.1X10~6 plates/m).A single DNA molecule observation during electrophoresis in a nanopillar channel revealed that the optimal nanopillars induced T4 DNA to form a narrow U-shaped conformation during electrophoresis whereas gamma DNA kepta a rather spherical conformation.We demonstrated that,even under a dc electric field,the optimal nanopillar dimensions depend on a gyration radius of DNA molecule that made it possible to separate large DNA fragments in a short time.