## 2 Materials and Methods
### On-Chip Chemical Syntheses of Reverse Oligonucleotides (5'-3')  
Solid-phase organic synthesis on a microchip has the advantages of a high reaction efficiency, low reagent consumption, and high product purity [14; 22]. Phosphoramidite chemistry was adopted for on-chip reverse chemical synthesis of oligos [23; 24]. Traditionally, oligonucleotide elongation is induced by repeating the four steps (deprotection, coupling, capping, oxidation, and four in-between washing steps) in a single cycle. A detailed scheme of the solid-phase reverse oligonucleotide synthesis on CPG as a solid support is shown in Figure 2. Before synthesis, glass vials (10 mL) were filled with 2 g, and molecular sieves were dried at 180 \({}^{\circ}\)C in a vacuum oven overnight to remove the absorbed moisture and other materials, if any, and were allowed to cool down to room temperature 15 min prior to use. All the used solutions were kept under a molecular sieve (3 A) for 24 h to make the solution completely moisture-free. Phosphoramidite compounds were dissolved in superdehydrated MeCN to form 0.1 mol/L solutions, and the CSO was dissolved in anhydrous MeCN (0.1 g/mL) and filtered through a 0.45 um filter. The porous CPGs were modified with the first nucleoside attached. First, the CPG particles were rinsed with anhydrous acetonitrile at a flow rate of 10 \(\upmu\)L min\({}^{-1}\) for 5 min to ensure continuous flow of the solvent through the chamber. The flow rate was optimized and maintained at 10 \(\upmu\)L min\({}^{-1}\) for all solutions. The first 3'-DMTr-protected nucleoside attached to the CPG particles was deprotected by introducing a mild deblocking reagent (5% trifluoroacetic acid (TFA) in anhydrous acetonitrile) at a flow rate of 10 \(\upmu\)L min\({}^{-1}\) for 2-4 min (the 5% TFA in acetonitrile shows high solvent compatibility with PDMS compared to conventional deblocking solvent dichloromethane (DCM), which swells the PDMS and destroys the microstructure by clogging the channel). The spectrophotometric assay of the resultant solution (containing trityl cations) was collected and measured to monitor the efficiency of the synthesis step. Stepwise coupling yields were estimated on the basis of the characteristic trityl absorbance assay in CH\({}_{2}\)Cl\({}_{2}\) (\(\epsilon=76000\) LM\({}^{-1}\)cm\({}^{-1}\)) (see supplementary information, 9) [25]. The residual deblocking reagent in the chamber was washed out through the outlet via a 2-3 min superdehydrated acetonitrile washing step performed at the same flow rate. Since the nitrogenous bases of the growing DNA chain are susceptible to acid-catalyzed deprivation, the deblocking step needs optimization, and an acetonitrile r  
Figure 2: Scheme of solid phase reverse synthesis of oligonucleotides on-chip using phosphoramidite chemistry. One cycle of single nucleotide addition is shown using circular arrows. The released dimethoxyttriyl (DMT) cation solution from the deprotection step was examined using absorbance for trekking the yield at each step. the deblocking agent from the support. Also, coupling efficiency and accuracy are increased by this wash, since premature detrtirylation of the incoming phosphoramidite monomer is prevented. To add the incoming monomer to the freed 3'-OH, the next phosphoramidite was first activated with the ETT activator for 30 s and then introduced into the chip from the inlet for 4 min. After the completion of the coupling reaction, the washing step was repeated for 2 min. Even with a high coupling efficiency, a small percentage of the 3'-OH group remained unreacted, which in subsequent coupling, led to deletion sequences. These active 3'-OH groups were inactivated with capping steps using cap mix A and cap mix B for 1 min, even though, in our study, we did not find any noticeable difference in the yield of the desired sequence with or without the capping step (confirmed with PAGE analysis, results are not shown here). A similar finding has been mentioned in a previous study [19]. Therefore, we eliminated the capping step from our synthesis cycle.