Large-scale microfabricated channel plates for high-throughput, fully automated DNA sequencing.
We have described a new DNA sequencing platform based on the Sanger chemistry, in which the large-scale microfabricated channel plates and electrophoretic system result in higher-throughput DNA sequencing. Three hundred and eighty-four channels are arranged in a fan-like shape on a 25x47 cm glass plate, on which 384 oval sample holes are connected to each channel coupled to the opposite anode access holes. Two microfabricated plates are set on the sequencing apparatus, in which sequencing electrophoresis is conducted on one plate and the preparation process is on another plate. Each sample hole is loaded with 2.3 microL volume of sample and injected into separation channels electrokinetically. High-quality sequencing data were acquired using the pUC18 template, achieving an average read-length of 1001 bases with 99% accuracy and a throughput of 5 Mbases per day per instrument. To assess the performance in actual sequencing field, the bacterial artificial chromosome shotgun library of the Pseudorca crassidens genome was sequenced, using 1/80 of the quantity of Sanger reagent (0.1 microL). We believe that this is the first demonstration of the useful performance of DNA sequencing using monolithic microfabricated devices with walk-away operation.
Kumagai
Hidesato
H
Shimadzu Corporation, Kyoto, Japan. kuma-h@shimadzu.co.jp
Utsunomiya
Shinichi
S
Nakamura
Shin
S
Yamamoto
Rintaro
R
Harada
Akira
A
Kaji
Toru
T
Hazama
Makoto
M
Ohashi
Tetsuo
T
Inami
Atsushi
A
Ikegami
Takashi
T
Miyamoto
Keisuke
K
Endo
Naoya
N
Yoshimi
Kenichi
K
Toyoda
Atsushi
A
Hattori
Masahira
M
Sakaki
Yoshiyuki
Y
D000818:Animals; D001483:Base Sequence; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004289:Dolphins / Q000235:genetics; D046209:Electrophoresis, Microchip / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D015723:Gene Library; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA / Q000295:instrumentation* / Q000379:methods
D004247:DNA