Photoelectromechanical synthesis of low-cost DNA microarrays

Abstract

Recent advances in de novo gene synthesis, library construction, and genomic selection for target sequencing using DNA from custom microarrays have demonstrated that microarrays can effectively be used as the world's cheapest sources of complex oligonucleotide pools. Unfortunately, commercial custom microarrays are expensive and not easily accessible to academic researchers, and technical challenges still exist for dealing with the small amount of DNA synthesized on a chip. Genomic research would certainly benefit from the creation of cheaper custom microarrays with larger oligonucleotide concentrations per spot. This thesis presents the development of a novel DNA microarray synthesis platform based on semiconductor photoelectrochemistry (PEC) designed with these needs in mind. An amorphous silicon photoconductor is activated by an optical projection system to create "virtual electrodes" that electrochemically generate protons in a site-selective manner, thereby cleaving acid-labile dimethoxytrityl protecting groups with the spatial selectivity that is required for in-situ DNA synthesis. This platform has the potential to be particularly low-cost since it employs standard phosphoramidite reagents, visible wavelength optics, and a cheaply microfabricated and reusable substrate. By incorporating a porous thin-film glass that dramatically increases the DNA quantity produced by over an order of magnitude per chip, this platform may also simplify the handling of DNA cleaved from chip and drive down the cost per base synthesized. The hybridization detection of single-base errors was successfully demonstrated on PEC synthesized microarrays. This thesis also reports a suite of new surface chemistries and high-resolution techniques for patterning biological molecules.