# Parallel DNA Synthesis to Produce Multi-Usage Two-Dimensional Barcodes
## 3 Results  
### Array Design, Principles and Synthesis  
The basic principle behind the design of a barcoded array is shown in Figure 1. The synthesizable area of a glass slide is first divided into two sections, one for the barcodes and one for the actual sequence library. The barcode area was a strip of \(\sim\)150 \(\times\) 768 pixels within a total area of 1024 \(\times\) 768 pixels, representing \(\sim\)15% of the synthesizable surface. The strip was chosen to host up to four different barcodes and test various barcoding strategies simultaneously while retaining sufficient space to synthesize a DNA library (Figure 1A). In reality, only a single barcode per array is necessary, which takes up a maximum of 150 \(\times\) 150 pixels, or \(<\)3% of the total synthesis area. The DNA library consists of a 97 mer sequence terminated with a 5\({}^{\prime}\) Cy3 dye and contains both forward and reverse primer binding regions embedded in the sequence (Figure 1B). Scanning the barcode in the barcode area reveals information on the primers to use and, therefore, on how to retrieve and amplify the DNA material recovered from the array (Figure 1C).  
The design and synthesis process of such a barcoded microarray is schematized in Figure 2. Two lists of DNA sequences, one for the DNA pool and one for the barcode strip, are used to populate the synthesis area (Figure 2A). A computer script assigns a position to each sequence and aims to recreate the digital barcode in scannable DNA format. Another script then transforms the sequence and location information into a series of instructions that allows for DNA synthesis in parallel using photolithography. The principle of DNA array photolithography has been described elsewhere [31], but briefly, for parallel DNA synthesis to occur, a photochemical reaction takes places only on the positions that are intended to receive the next nucleotide. This photochemical reaction removes a 5\({}^{\prime}\) photoprotecting group, releasing 5\({}^{\prime}\)-OH that can react with the next base in the form of DNA phosphoramidite. The selective illumination of the desired locations on the surface of the array employs micromirrors that can reflect light (here, 365 nm UV light) onto or away from the surface. Our current photolithography setup is equipped with an  
Figure 1: **(A) Microarray subdivision of the addressable area into areas for library synthesis and for barcode synthesis. The barcode area is a strip of \(\sim\)150 \(\times\) 768 pixels, or \(\sim\)2.1 \(\times\) 10.7 mm\({}^{2}\) given the size of a single addressable unit (14 \(\times\) 14 \(\upmu\)m\({}^{2}\)), and contains up to four DNA barcodes (QR codes or data matrix). (B) The DNA library synthesized in the library area is a 97 mer with both forward and reverse primers (in italic) synthesized along with the insert. A Cy3 dye terminates the strand at the 5\({}^{\prime}\) end. (C) An example of a minimal, digitally created QR code delivering primer sequence information upon scanning and thus allowing for the amplification of the synthesized DNA.**  
array of 1024 \(\times\) 768 such micromirrors, 14 \(\upmu\)m in size, making a single micromirror the equivalent of a synthesizable pixel. DNA photolithography was carried out in order to prepare the DNA library and the DNA barcodes at the same time. Standard photoprotected DNA phosphoramidites (Figure 2B) were used to grow DNA oligonucleotides. To cleave the DNA library and to effect a loss of Cy3 fluorescence in the barcode area, cleavable oligonucleotides were synthesized atop a succinyl-dT unit, while fluorescent tagging employed a Cy3 phosphoramidite as the last coupling step.