Optimization of DNA microarray printing

Abstract

DNA microarrays are often used in the detection and characterization of microbial pathogens in clinical applications. Their use combines molecular biology, robot dispensing technology, and readout applications. Molecular biology is needed in preparing and modifying the DNA and microarray surfaces for printing and in hybridization of samples to the microarray. Robot dispensing technologies are used to produce small (typically a few picolitres) spots of DNA fragments on the microarrays. Readout applications are then used to locate the spots and detect the emission of fluorescence from the microarray.

This thesis was a part of a project conducted at the Mobidiag Research & Development department in Espoo, Finland. The purpose of this thesis was to optimize DNA microarray printing and to explore and create the possibility of microarray printing in the company’s laboratories using devices that dispense larger droplets. Two different liquid handlers, which can dispense droplets of a few nanoliters, were selected for the testing because they would suit the needs of the research and development laboratories for fast printing of different types of microarrays on 96-well plates. The aim of this study was to test how larger droplet volumes effect the performance of microarrays. The tests were performed using different droplet volumes, pitches, and layouts and 96-well plates to obtain good quality microarrays for research use.

Tested droplet volumes were 2.5, 7.5, and 20 nanoliters. The pitch between spots was optimized to be between 0.4 – 1 millimeter depending on the droplet volume. The size of the arrays was between 7x7 and 4x4 to ensure that the spots fit into the well bottoms. Each volume was tested to see if it causes cross-reactivity or affects the performance of the microarray by disturbing its readability. The experiments showed that the droplet volume had no effect on the performance of the microarray when the pitch and layout were adjusted accordingly. The arrays were also tested for their specificity and all selected targets performed correctly. Different DNA concentrations were tested on the spots and hybridization buffer mixes together with different droplet volumes to evaluate the effects on the performance of microarrays. It was concluded that the starting DNA-concentration (20 µM) could be lowered for larger volumes without any effect on the readouts.