Improvement Of A Sequential Injection Microfluidic Pyrosequencing System And Its Application

DNA sequencing technology is one of the core technologies of life science research. Pyrosequencing is a real-time DNA sequencing method, which is fast, efficient and low cost, comparing to that based on Sanger reaction. There are two main trends for pyrosequencing: one is high throughput system for large scale genome sequencing, the other is small or portable system for point of care test purpuses. In this paper, the established bead-based micro-reactor microfluidic pyrosequencing system was improved, and demonstated by swine influenza virus H1N1MP gene typing and single nucleotide polymorphism (SNP) assay.Firstly, the magnetic beads (MB) bearing streptavidin were fixed in a capillary by permanent magnets through two pieces of silicon-steel sheet, and the MBs were made vibrating periodically by changing the position of the capillary between the two sheets. The extension reaction rate was effectively improved, which is crucial for a success flow sequencing. The microfluidic flow reaction system is easy to establish without involveing any micro-fabrication.By remote magnetic control, fixing, replacing and vibrating of the MBs are easily manipulated. Combined with sequential injection of reactive reagents, flexible microfluidic flow analysis platform was established.Secondly, a microfluidic flow sequential injection pyrosequencing system was constructed based on the above method, using a weak chemiluminescence meter as detector, a syringe injection pump as the driving force and a slotted microvial array as the reagent delivering system. By adding apyrase to the reaction solution, the background signal of the three-enzyme system were greatly reduced. The reaction conditions, including flow rate capillary diameter were studied systematically. Ultimately, the whole sequence of a synthetic single-strand DNA (28base pairs) was read with good agreement. The applicability was demonstrated by a H1N1virus MP gene sequencing and a SNP typing, and the results were generally consistent with that obtained by a well established static liquid pyrosequencing system.This microfluidic pyrosequencing system is simple, cost-effective and easy to realize portability and automation. Through further improvement in the repeating bases reading, this system is very promising in overcoming the read length limit of pyrosequencing.