Development Of Fast Electrophoretic Separation Systems For DNA Fragments Based On A Microfluidic Chip And A Short Capillary

Capillary gel electrophoresis (CGE) has become an efficient tool for separation of DNA restriction fragments, PCR products and DNA sequencing. In conventional CGE systems, separation of DNA is usually performed in a capillary with a separation length of 20-100 cm, inner diameter in the range of 50-100μm. The separation time is among 10-30 min. However, the expensive equipments and complex operation limit its widely application in most biology laboratory. Actually, traditional slab gel electrophoresis is still used for DNA analysis in most laboratories. Therefore, developing a low-cost, effective and fast DNA separation system is still desirable.Since the introduction of high speed capillary electrophoresis (HSCE) in the 1990s, microfluidic chip has offered a promising way to achieve high-speed DNA separation. Some microfluidic chip-based commercial instruments for DNA separation have been released in the market. However, fast DNA separation can also be achieved in a short capillary, which avoids the complex fabrication and operation in chip-based systems.In chapter 1, the mechanism of DNA separation and progress of fast DNA separation systems based on conventional CGE mode, microfluidic chip and short capillary were reviewed. The recent progress of commercial integrated microfluidic chip-based DNA analyzer was also introduced.In chapter 2, an integrated DNA analyzer involving microfluidic chip, high voltage power supply, laser-induced fluorescence (LIF) detection system and control system was developed to perform fast CE separation. An orthogonal optical arrangement was employed in the LIF detection system to simplify the structure of the system with a limit of detection of 1.0x10-10 mol/L for Cy5 dye. A simple and convenient approach for accurately locating the chip in the DNA analyzer was developed by using a chip supporter and base pegs with a chip position precision of±2μm. The performance of the present DNA analyzer was demonstrated in the separation ofΦX174-HaeⅢdigest DNA Marker.In chapter 3, a fast DNA fragment separation system was developed based on a short capillary and a slotted-vial-array automated sample introduction system. The injection process of DNA sample in a short capillary were investigated systematically with three injection techniques including constant-field-strength, low-field-strength and translational spontaneous injections. Under the optimized conditions, picoliter-scale sample plugs (corresponding to ca.20-μm plug length) were obtained, which ensure the high-speed and high-efficiency separation for DNA fragments with a short effective separation length. Other separation conditions including the sieving matrix concentration, separation field strength and effective separation length were also optimized. The present system was applied in the separation of OX174-HaeⅢdigest DNA marker. With an effective separation length of 2.5 cm, the separation could be achieved in less than 100 s with plate heights ranging from 0.21 to 0.74μm (corresponding to plate numbers from 4.86x106 to 1.36x106 per meter). The repeatabilities for the migration time of the 11 fragments were between 0.4%-1.1% RSD (n=8). By using the automated continuous injection method, the separation for four different DNA samples could be achieved within 250 s. The present system was further applied in the fast sizing of real DNA samples of PCR products.In chapter 4, a simple fabrication method of tapered capillary tip was developed using grind method by sand papers. The controllability to the angle and end area of the tapered tip end of the capillary was significantly improved by using the self-elasticity of capillary and its regular precession. Compared with the conventional capillary tip fabrication methods, the present method has advantages of easy to use without the need of expensive equipments or potentially dangerous HF etching. Potential application of the tapered capillaries fabricated by this method in the high-speed capillary electrophoresis (CE) and eletrospray ionization mass spectrometry (ESI-MS) was demonstrated.