DNA sequencing on microfabricated electrophoretic devices.
We present a model that quantitatively describes the performance of microfabricated electrophoretic devices filled with linear polyacrylamide as replaceable sieving material for single-stranded DNA analyses. The dependence of resolution on various separation parameters such as selectivity, diffusion, injector size, device length, and channel folding was investigated. A previously predicted dependence of longitudinal diffusion coefficient on electric field strength has been verified. We have used this model to develop and optimize microfabricated electrophoretic devices for DNA analyses. For single-color DNA sequencing mixtures, we routinely achieve separations of 400 bases in under 14 min at 200 V/cm, and separation of 350 bases in only 7 min at 400 V/cm, with a minimum resolution of R = 0.5. Our results also indicate reduced fragment biasing and efficient sample stacking for DNA sample loading on microfabricated devices.
Schmalzing
D
D
Whitehead Institute for Biomedical Research, Nine Cambridge Center, Massachusetts 02142, USA.
Adourian
A
A
Koutny
L
L
Ziaugra
L
L
Matsudaira
P
P
Ehrlich
D
D
D000180:Acrylic Resins / Q000737:chemistry; D053938:DNA Fragmentation; D004277:DNA, Single-Stranded / Q000032:analysis* / Q000737:chemistry*; D004058:Diffusion; D004574:Electromagnetic Fields; D019075:Electrophoresis, Capillary / Q000379:methods*; D012822:Silicon Dioxide / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D000099166:Polyacrylamides
D000180:Acrylic Resins; D004277:DNA, Single-Stranded; D012822:Silicon Dioxide; D000099166:Polyacrylamides