Tomoyuki Sakai | PubMed Health

PubMed2006

Ultra-slim laminated capillary array for high-speed DNA separation.

We developed a new kind of capillary array for electrophoresis by using the numerical-control (NC) wiring technique conventionally used to produce printed-circuit boards. Laminating two polyimide sheets after laying cylindrical capillaries between them according to designed geometries, we fabricated a 16-lane laminated capillary array (LCA) 9.9 cm long, 7.2 cm wide, and 0.5 mm thick in which the effective length of all capillaries was only 10.9 cm. This compact LCA thus had separation columns as short as those in capillary array electrophoresis chips fabricated by lithography techniques. Like conventional capillary arrays, it also enabled pipetting-less direct injection of analytes from sample preparation plates. Using the LCA with LIF detection and a replaceable fluid sieving matrix, we demonstrated high-speed ssDNA fragment separations. At an electric field strength of 316 V/cm, 15 fragments ranging from 50 to 500 bases were completely separated within 5.8 min in all lanes. The lane-to-lane CV of migration time was only 0.38%, and the fragment size for which the resolution per base was 0.59 was 258 +/- 15 bases (average +/-SD).

DNA, Single-Stranded↗

PubMed2004

High-speed DNA sequencing by tube-based capillary electrophoresis.

We assessed the feasibility of high-speed DNA sequencing by tube-based capillary electrophoresis (TCE) with electrokinetic sample injections. We developed a water-circulated TCE system to control the capillary temperature precisely. Using this system and a ready-made sieving matrix at 50 degrees C, single-stranded DNA size marker fragments were separated at various pairs of the electric field strength, E, of 128-480 V/cm and the capillary effective length, L, of 100-360 mm. Assuming the read length (RL) is the fragment size at which the peak width equals the peak interval per base in obtained electropherograms, we estimated the values of RL (E, L), the RL at the pair (E, L). The points in ELz-space, (E, L, RL(E, L)), form a curved surface expressed by z = RL(E, L). Analyzing the contour lines of this curved surface, we determined the pairs of E and L providing target RLs of 300-500 bases within a minimum time. At a pair optimized for a 500-base RL (330 V/cm, 200 mm), one-color sequencing fragments were successfully separated up to 529 bases within 9.6 min. These results demonstrate that high-speed DNA sequencing comparable with that obtained by microfabricated chip-based capillary electrophoresis (MCE) can be achieved with TCE, which is more suitable in automation than MCE.

Electrophoresis, Capillary↗