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.
Sakai
Tomoyuki
T
Hitachi Ltd., Central Research Laboratory, Tokyo, Japan. sakai-t@crl.hitachi.co.jp
Sonehara
Tsuyoshi
T
Goda
Chifumi
C
Kohara
Yoshinobu
Y
Anazawa
Takashi
T
D019075:Electrophoresis, Capillary / Q000295:instrumentation* / Q000379:methods; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*