Silica Nanoparticles As Adsorbent For Solid-phase Extraction Of Genomic DNA From Human Whole Blood

Purification of samples is of vital importance for most DNA analysis because of complex matrix interference. In the present work, a rapid separation and purification method with high efficiency using silica nanoparticles as a new adsorbent for DNA solid-phase extraction was developed, and the on-line extraction of genomic DNA from human whole blood was accomplished.In Chapter1, the significance, development and popular methods of DNA separation and purification were reviewed. Besides, the working principle and common adsorbents for DNA solid-phase extraction (SPE), on-line DNA SPE techniques, as well as the development of nano materials as adsorbents for solid-phase extraction were described. At last, the consideration of our work design was given.In Chapter2, a DNA separation and purification method using silica nanoparticles as a new adsorbent was established. DNA adsorption mechanism was studied. The effects of the guanidine hydrochloride concentration, the pH of DNA sample, the adsoption time, the adsorption temperature, the pH of elution solution, the elution time and the elution temperature on the recovery efficiency of DNA have been investigated.98%DNA was adsorbed to the surface of silica nanoparticles within0.5min. The DNA adsorption capacity of silica nanoparticles was124ng/mg on average. When eluted by1×TE (pH8.3) for15min at55℃, a DNA recovery efficiency of90%was achieved. The whole extraction procedure was accomplished within25min.In Chapter3, a sequential injection on-line SPE system was developed for genomic DNA purification from human whole blood. The extraction column was heated to improve the DNA recovery. The effects of sample loading flow rate, elution flow rate and temperature on DNA recovery efficiency were investigated. Under the optimized conditions, DNA was successfully extracted with an overall recovery efficiency of69%, and the whole extraction process takes less than4min. The subsequent PCR amplification indicated that this system was qualified for DNA purification from human whole blood samples.In the last chapter, a summary and perspective were given for this rapid and highly efficient DNA separation and purification system.