On-line nanoliter cycle sequencing reaction with capillary zone electrophoresis purification for DNA sequencing.
An integrated system for DNA sequencing based on a nanoreactor for cycle-sequencing reaction coupled with on-line capillary zone electrophoresis (CZE) for purification and capillary gel electrophoresis (CGE) for separation is presented. Less than 100 nl of premixed reagent solution, which includes dye-labeled terminator pre-mix, bovine serum albumin and template, was hydrodynamically injected into a fused-silica capillary (75 microm I.D.) inside a laboratory-made microthermocycler for cycle sequencing reaction. In the same capillary, the reaction products were purified by CZE followed by on-line injection of the DNA fragments into another capillary for CGE. Over 540 base pairs (bp) of DNA can be separated and the bases called for single-standed DNA with 0.9% error rate. The total time was about 3.5 h, or a cycle time of 2 h with staggered operation. For double-stranded DNA, a longer reaction time was required and base calling up to 490 bp with 1.2% error rate was achieved. The whole system is readily adaptable to automated multiplex operation for DNA sequencing or polymerase chain reaction analysis.
Xue
G
G
Iowa State University, Ames Laboratory-US Department of Energy and Department of Chemistry, 50011, USA.
Pang
H M
HM
Yeung
E S
ES
D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods*; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA / Q000379:methods*; D013050:Spectrometry, Fluorescence
D004247:DNA