On-line nanoliter cycle sequencing reaction with capillary zone electrophoresis purification for DNA sequencing

• Published in: Journal of Chromatography A 2001-04, Vol.914 (1), p.245-256
• Main Authors: Xue, Gang, Pang, Ho-Ming, Yeung, Edward S
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Base Sequence; Biological and medical sciences; DNA; DNA - chemistry; Dna, deoxyribonucleoproteins; Electrophoresis, Capillary - methods; Fundamental and applied biological sciences. Psychology; Molecular Sequence Data; Nucleic acids; Sequence Analysis, DNA - methods; Spectrometry, Fluorescence
• ISSN: 0021-9673
• DOI: 10.1016/S0021-9673(00)01167-5

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 μm 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.