In situ Synthesis of DNA Microarray on Functionalized Cyclic Olefin Copolymer Substrate

Abstract

Thermoplastic materials such as cyclic-olefin copolymers (COC) provide a versatile and cost-effective alternative to the traditional glass or silicon substrate for rapid prototyping and industrial scale fabrication of microdevices. To extend the utility of COC as an effective microarray substrate, we developed a new method that enabled for the first time in situ synthesis of DNA oligonucleotide microarrays on the COC substrate. To achieve high-quality DNA synthesis, a SiO 2 thin film array was prepatterned on the inert and hydrophobic COC surface using RF sputtering technique. The subsequent in situ DNA synthesis was confined to the surface of the prepatterned hydrophilic SiO 2 thin film features by precision delivery of the phosphoramidite chemistry using an inkjet DNA synthesizer. The in situ SiO 2 -COC DNA microarray demonstrated superior quality and stability in hybridization assays and thermal cycling reactions. Furthermore, we demonstrate that pools of high-quality mixed-oligos could be cleaved off the SiO 2 -COC microarrays and used directly for construction of DNA origami nanostructures. It is believed that this method will not only enable synthesis of high-quality and low-cost COC DNA microarrays but also provide a basis for further development of integrated microfluidics microarrays for a broad range of bioanalytical and biofabrication applications.

Error correction of microchip synthesized genes using Surveyor nuclease

“…Oligonucleotides were synthesized on a plastic chip using a custom-made inkjet DNA microarray synthesizer (32). Gene-construction oligos were designed to be 60-nt long with overlapping regions of similar melting temperatures ( T m  = 65 ± 2°C).…”

Polymeric-Based In Vitro Diagnostic Devices

“…Another vitally important property of COP is its low autofluorescence, which lowers the background noise when fluorescence detection is employed. The autofluorescence of COP is higher than that of glass or PDMS, but in the same order of magnitude as that of PMMA or PC [33,35].…”

Synthetic DNA Synthesis and Assembly: Putting the Synthetic in Synthetic Biology

“…Edge effects reduce oligo quality by reducing the selective control of the synthesis reactions in adjacent oligonucleotides, which can lead to substitution or deletion errors in oligonucleotides synthesized on adjacent features. Work to reduce edge effects during the synthesis process has shown that refinements to the chip design can improve the fidelity of array-synthesized oligonucleotides to rival column-synthesized oligonucleotides (Saaem et al 2010). Continued improvement in array design and refinements to synthesis reagents and processes used will lead to routine robust synthesis of high-quality long oligonucleotides from arrays, which will further their use as the go to source for low-cost oligonucleotides for gene synthesis applications.…”