Automated DNA fragment collection by capillary array gel electrophoresis in search of differentially expressed genes.
An automatic DNA fragment collector using capillary array gel electrophoresis has been developed. A sheath flow technique is used for not only detection but also collection of DNA fragments. In a sheath flow cell, the DNA fragments separated by 16 capillaries flow independently into corresponding sampling capillaries. The fraction collector consists of 16 sampling trays and each sampling tray is set beneath each end of the sampling capillaries to collect the flow-through DNA fragments. Certain DNA fragments are automatically sorted by controlling the movement of the sampling trays according to the signals from the system. The collector experimentally separated two mixtures of polymerase chain reaction (PCR) products: one prepared by using eight different sizes (base lengths from 161 to 562) of DNAs; and the other prepared by a differential display (DD) method with cDNA fragments. Collected DNA fragments are amplified by PCR and measured by electrophoresis. DNA fragments with base length differences of one (base lengths 363 and 364) were successfully separated. A separated DNA fragment from the DD sample was also successfully sequenced. In addition, differentially expressed DNA fragments were automatically sorted by comparative analysis, in which two similar cDNA fragment groups, labeled by two different fluorophores, respectively, were analyzed in the same gel-filled capillary. These results show that the automatic DNA fragment collector is useful for gene hunting in research fields such as drug discovery and DNA diagnostics.
Irie
T
T
Central Research Laboratory, Hitachi, Kokubunji, Tokyo, Japan. tak-irie@crl.hitachi.co.jp
Oshida
T
T
Hasegawa
H
H
Matsuoka
Y
Y
Li
T
T
Oya
Y
Y
Tanaka
T
T
Tsujimoto
G
G
Kambara
H
H
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000032:analysis* / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods*; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*
D004247:DNA