High-speed DNA genotyping using microfabricated capillary array electrophoresis chips.
Capillary array electrophoresis (CAE) chips have been designed and fabricated with the capacity to rapidly (< 160 s) analyze 12 different samples in parallel. Detection of all lanes with 0.3 s temporal resolution was achieved using a laser-excited confocal-fluorescence scanner. The operation and capabilities of these CAE microdevices were first determined by performing electrophoretic separations of pBR322 MspI DNA samples. Genotyping of HLA-H, a candidate gene for the diagnosis of hereditary hemochromatosis, was then performed to demonstrate the rapid analysis of biologically relevant samples. Two-color multiplex fluorescence detection of HLA-H genotypes was accomplished by prelabeling the standard pBR322 MspI DNA ladder with a red emitting bis-intercalation dye (butyl TOTIN) and on-column labeling of the HLA-H DNA with thiazole orange. This work establishes the feasibility of using CAE chips for high speed, high-throughput genotyping.
Woolley
A T
AT
Department of Chemistry, School of Public Health, University of California, Berkeley 94720, USA.
Sensabaugh
G F
GF
Mathies
R A
RA
D052160:Benzothiazoles; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D053938:DNA Fragmentation / Q000235:genetics; D019075:Electrophoresis, Capillary; D005456:Fluorescent Dyes / Q000737:chemistry; D005838:Genotype; D006680:HLA Antigens / Q000235:genetics*; D006432:Hemochromatosis / Q000235:genetics*; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I / Q000235:genetics*; D006650:Histocompatibility Testing / Q000379:methods; D006801:Humans; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D011804:Quinolines; D013844:Thiazoles / Q000737:chemistry
D052160:Benzothiazoles; D005456:Fluorescent Dyes; C103673:HFE protein, human; D006680:HLA Antigens; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I; D008565:Membrane Proteins; D011804:Quinolines; D013844:Thiazoles; C051000:thiazole orange; D004247:DNA