Abstract
BackgroundDNA 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.ResultsHigh 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.ConclusionsHighly 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.
Highlights
DNA microarrays are a core element of modern genomics research and medical diagnostics, allow‐ ing the simple and simultaneous determination of the relative abundances of hundreds of thousands to millions of genomic DNA or RNA sequences in a sample
We have successfully used 4,5-dicyanoimidazole (DCI) [32] as an activator in microarray synthesis for many years, but decided to test alternative activators that might allow for faster coupling
One of the key differences between standard solid-phase synthesis of oligonucleotides and photolithographic synthesis is that solid-phase synthesis relies on the use of the acid-labile dimethoxytrityl (DMT) 5′-hydroxyl protecting group, and is somewhat sensitive to very acidic activators, which can prematurely remove some DMT groups, leading to n+1 errors [33]
Summary
DNA microarrays are a core element of modern genomics research and medical diagnostics, allow‐ ing the simple and simultaneous determination of the relative abundances of hundreds of thousands to millions of genomic DNA or RNA sequences in a sample. The photolithographic method is based on the use of optical imaging systems to deliver light to the synthesis surface, where array layout and sequences are determined by selective removal of the photocleavable protecting groups on the terminus of each oligonucleotide. The flexibility of the approach results with the use of an imaging system centered on a digital micromirror device (DMD) in place of photomasks to deliver patterned light to the synthesis surface This approach, termed Maskless Array Synthesis (MAS), allows virtual masks to control the layout and the oligonucleotide sequences on the array. The speed of the photolithographic approach is due to the ability to use high efficiency photolabile groups that can be removed quickly with light exposure, and the minimal set-up time for new microarray designs. The optimization experiments presented here include the evaluation of alternative activators and activator concentrations, the determination of the optimal coupling time, the best oxidation strategy, and other chemical synthesis parameters
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