Optimization of high-performance DNA sequencing on short microfabricated electrophoretic devices.
We have examined the parametric performance of short microfabricated electrophoresis devices that operate with a replaceable linear poly(acrylamide) (LPA) solution for the application of DNA sequencing. A systematic study is presented of the dependence of selectivity, separation efficiency, and resolution of sequencing fragments on buffer composition, LPA concentration, LPA composition, microdevice temperature, electric field, and device length. A specific optimization is made for DNA sequencing on 11.5-cm devices. Using a separation matrix composed of 3.0% (w/w) 10 MDa plus 1.0% (w/w) 50 kDa LPA, elevated microdevice temperature (50 degrees C), and 200 V/cm, high-speed DNA sequencing of 580 bases on standard M13mp18 was obtained in only 18 min with a base-calling accuracy of 98.5%. Read lengths of 640 bases at 98.5% accuracy were achieved in approximately 30 min by reducing the electric field strength to 125 V/cm. We believe that this constitutes matrix-limited performance for microdevices of this length using LPA sieving matrix and this buffer chemistry. In addition, it was confirmed, that shorter devices are rather impractical for production sequencing applications when LPA is used as sieving matrix.
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA.
O
O
Salas-Solano
D
D
Schmalzing
L
L
Koutny
S
S
Buonocore
A
A
Adourian
P
P
Matsudaira
D
D
Ehrlich
D001483:Base Sequence; D002021:Buffers; D004247:DNA / Q000032:analysis*; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA / Q000379:methods*
D002021:Buffers; D004247:DNA