High-Efficiency Reverse (5′→3′) Synthesis of Complex DNA Microarrays

DNA microarrays are important analytical tools in genetics and have recently found multiple new biotechnological roles in applications requiring free 3′ terminal hydroxyl groups, particularly as a starting point for enzymatic extension via DNA or RNA polymerases. Here we demonstrate the highly efficient reverse synthesis of complex DNA arrays using a photolithographic approach. The method is analogous to conventional solid phase synthesis but makes use of phosphoramidites with the benzoyl-2-(2-nitrophenyl)-propoxycarbonyl (BzNPPOC) photolabile protecting group on the 3′-hydroxyl group. The use of BzNPPOC, with more than twice the photolytic efficiency of the 2-(2-nitrophenyl)-propoxycarbonyl (NPPOC) previously used for 5′→3′ synthesis, combined with additional optimizations to the coupling and oxidation reactions results in an approximately 3-fold improvement in the reverse synthesis efficiency of complex arrays of DNA oligonucleotides. The coupling efficiencies of the reverse phosphoramidites are as good as those of regular phosphoramidites, resulting in comparable yields. Microarrays of DNA surface tethered on the 5′ end and with free 3′ hydroxyl termini can be synthesized quickly and with similarly high stepwise coupling efficiency as microarrays using conventional 3′→5′ synthesis.© The Author(s) 201

Express photolithographic DNA microarray synthesis with optimized chemistry and high-efficiency photolabile groups

Background: DNA microarrays are a core element of modern genomics research and medical diagnostics, allowing the simple and simultaneous determination of the relative abundances of hundreds of thousands to millions of genomic DNA or RNA sequences in a sample. Photolithographic in situ synthesis, using light projection from a digitally-controlled array of micromirrors, has been successful at both commercial and laboratory scales. The advantages of this synthesis method are its ability to reliably produce high-quality custom microarrays with a very high spatial density of DNA features using a compact device with few moving parts. The phosphoramidite chemistry used in photolithographic synthesis is similar to that used in conventional solid-phase synthesis of oligonucleotides, but some unique differences require an independent optimization of the synthesis chemistry to achieve fast and low-cost synthesis without compromising microarray quality. Results: High microarray quality could be maintained while reducing coupling time to a few seconds using DCI activator. Five coupling activators were compared, which resulted in microarray hybridization signals following the order ETT > Activator 42 > DCI ≫ BTT ≫ pyridinium chloride, but only the use of DCI led to both high signal and highly uniform feature intensities. The photodeprotection time was also reduced to a few seconds by replacing the NPPOC photolabile group with the new thiophenyl-NPPOC group. Other chemical parameters, such as oxidation and washing steps were also optimized. Conclusions: Highly optimized and microarray-specific phosphoramidite chemistry, along with the use of the very photosensitive thiophenyl-NPPOC protecting group allow for the synthesis of high-complexity DNA arrays using coupling times of 15 s and deprotection times of 9 s. The resulting overall cycle time (coupling to coupling) of about 50 s, results in a three-fold reduction in synthesis time

Hamming Distance as a Concept in DNA Molecular Recognition

DNA microarrays constitute an in vitro example system of a highly crowded molecular recognition environment. Although they are widely applied in many biological applications, some of the basic mechanisms of the hybridization processes of DNA remain poorly understood. On a microarray, cross-hybridization arises from similarities of sequences that may introduce errors during the transmission of information. Experimentally, we determine an appropriate distance, called minimum Hamming distance, in which the sequences of a set differ. By applying an algorithm based on a graph-theoretical method, we find large orthogonal sets of sequences that are sufficiently different not to exhibit any cross-hybridization. To create such a set, we first derive an analytical solution for the number of sequences that include at least four guanines in a row for a given sequence length and eliminate them from the list of candidate sequences. We experimentally confirm the orthogonality of the largest possible set with a size of 23 for the length of 7. We anticipate our work to be a starting point toward the study of signal propagation in highly competitive environments, besides its obvious application in DNA high throughput experiments.Copyright © 2017 American Chemical Societ