A Capillary Array Gel Electrophoresis System Using Multiple Laser Focusing for DNA Sequencing

Abstract

A very simple and highly sensitive capillary array gel electrophoresis system is constructed to analyze DNA fragments. On-column detection of DNA migration in a large number of gel-filled capillaries is carried out using side-entry laser irradiation and with a CCD camera, although it has been considered impossible because the irradiation laser is scattered strongly at the surfaces of the first few capillaries. By optimizing optical conditions, the laser beam can be focused repeatedly to irradiate all the capillaries held on a plate by working each capillary as a cylindrical convex lens. DNA sequencing samples migrating in 24 capillaries can simultaneously be analyzed with the system.

A capillary-array electrophoresis system using side-entry on-column laser irradiation combined with glass rod lenses

“…The fluorescence-detection system was the same as reported previously [15]. Fluorescence from all the capillaries was detected simultaneously with a CCD camera and the prism system provided color selectivity.…”

“…The laser beam is repeatedly focused by the convex lens action of each capillary and passes through all the capillaries (multiple-laser focusing). The focusing action of each capillary is expressed by the refraction angle, Dq, which is defined as the angle between the incident laser beam and the refracted laser beam passing through a capillary [15]. When a laser beam of an infinitesimal diameter enters a capillary at a distance of r/2 from the center of the capillary, the refraction angle is given by where R and r are the outer and inner radii of a capillary, respectively, and n 1 , n 2 , and n 3 are the refractive indexes of the outside, capillary-wall, and inside mediums, respectively.…”

“…We [15] and other groups [14,16] have developed capillary-array systems that use both side-entry laser irradiation and on-column detection. In the original configuration of our system, all 24 capillaries were placed in air.…”

“…Figure 1 shows a schematic view of our capillary-array assembly and fluorescence detection system. Preparation of the capillary gel, consisting of a 4%T, 5%C polyacrylamide gel, containing 1 TBE (89 mM Tris 89 mM boric acid and 2.5 mM EDTA, pH 8.3) and 7 M urea, was based on the same method as reported previously [15]. The total and effective lengths of each capillary were set as 40 and 25 cm, respectively.…”

Construction and evaluation of a capillary array DNA sequencer based on a micromachined sheath-flow cuvette

“…The high temperature relaxes intrastrand hydrogen bonds, eliminating sequencing artifacts due to compression. Kambara and Takahashi [20] and Kambara and Takahashi and co-workers [21,22] produced a 20-capillary array instrument with a sheath-flow detection cell. In this system, a set of receiving capillaries, coaxial with the separation capillaries, received the sample stream and sheath fluid.…”