Direct PCR From Plasma On Chip And Establishment Of A Microfluidic Chemiluminescence SNP Analytical System

DNA is an important kind of biological macromolecules, and its analysis is receiving widespread attention. Improvements of DNA analysis depend very much on method development, and miniaturization and integration are two of these trends. The appearance of micro total analysis system (Micro-TAS) is also a reflection of the development. Blood is a typical and commonly used clinical sample, and plasma DNA is cell-free DNA in the blood. Beacause of its unique significance in disease detection and diagnosis, plasma DNA ananlysis is of prime interest both in medicine and scientific research. In this thesis, a direct plasma DNA PCR amplification method was developed, and implemented on a static chip PCR system; Furthermore, a microfluidic chemiluminscence DNA analysis system was established, and a SNP typing was demonstrated with the system.Firstly, direct plasma DNA PCR amplification on a static chip thermostat without purification procedure was investigated. The amplification conditions such as the composition of reagents and thermal programs were studied systematically with a GeneAmp PCR system with an exteriorλDNA segment (236bp) as the target, and the method was applied for DYZ-1 gene (149bp) amplification directly from plasma samples. Satisfactory results were obtained with plasma sample ranging from 0.1μL to 16.7μL within a 25μL PCR reaction system. Direct amplifications of exterior X-DNA and plasma DYZ-1 gene were successfully demonstrated by a static chip PCR as characterized by gel electrophoresis and confirmed by PCR-chip electrophoresis.Secondly, a simple microfluidic chemilluminscence DNA analyzing system was established based on pyro-sequencing reactions. Various forms of microreactors were investigated for immobilizing the magnetic beads bearing the template, incluiding a glass HGMS (high gradient magnetic seperation) chip, a PDMS-ITO glass HGMS chip and a capillary chip. Fused silica capillary was chosen both for reagent infusion and reaction cell, and the bead templates were fixed by permanet magnets on both sides under attraction mode. A iron shielding box was used avoiding possible harm effect of magnetic to the photo multiplier. SNP typing of rs8130833 on chromosome 21 was achived taking advantage of BAMPER (Bioluminometric Assay coupled with Modified Primer Extension Reactions). Comparing to the static method where 4 enzymes were involved, this method is simpler, faster and more reagent saving.