A Spatial Temperature Gradient CE System With Automated Sample Introduction For Single Nucleotide Polymorphisms Detection

Gene mutation is closely related with many diseases, such as diabetes, cancer, heart disease, etc. Single nucleotide polymorphisms (SNPs) is one of the most common gene mutation. Temperature gradient gel electrophoresis (TGGE) is an ideal method for SNPs detection, and gene fragments with various sizes or different mutations can be assayed under the same temperature gradient. In this paper, an automated high-throughput capillary electrophoresis system with a simple, reliable, convenient spatial temperature gradient device and a continuous sample introduction device based on a slotted vial array for SNPs detection was developed.In Chapter 1, the recent progress in SNPs detection techniques of single-strand conformational polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), conformation sensitive gel electrophoresis (CSGE), TGGE and denaturing high performance liquid chromatography (DHPLC) was reviewed. The development of formation methods of temperature gradient in TGGE and sample introduction methods of microfluidic capillary electrophoresis (CE) was also introduced.In Chapter 2, a spatial temperature gradient capillary electrophoresis system with automated sample introduction for SNPs detection was established. An automated slotted vial array sample introduction device was combined to the capillary electrophoresis system, so that continuous electrokinetic sample injection can be achieved. The temperature gradient was produced by a radiative heating system with a graphite block heater, and the stability of the temperature gradient was investigated. The temperature variation of each measure point was 0.12-0.21% RSD within 6 h. A 14-cm Teflon AF coated silica capillary was used as separation channel and waveguide tube of fluorescene signal. The concentration and the type of sieving matrix, the pH and the type of buffer, field strength were optimized. Under the same temperature gradient, all 4 fragments of a Low-range control mutation standard (209 bp) were separated with analytical time of 4 min and a carryover of 3.3%, and K-ras gene mutations from a patient with colorectal cancer were successfully detected.In Chapter 3, the spacial temperature gradient capillary electrophoresis system with automated sample introduction was summarized.