Light directed deprotection method.
The production of oligonucleotide microarrays makes use of a combination of photolithography with DNA-synthetic chemistry. In this technique, nucleotides are used in the form of modified phosphoramidites (as used in DNA synthesis machines), having a photo-labile protecting group, so that these protecting groups can be removed by exposing nucleotides to light. In other words, light is used as an activating agent in the synthesis reaction. Photolithographic masks are used to control the regions of the chip, that are illuminated but not desired to be deprotected. After deprotection the surface of the chip is flooded with one of the four bases to allow selective coupling of that base to each deprotected region of the synthesis surface. A seconded region of the chip is then deprotected and similarly flooded with another

base for coupling reaction. In this manner, four cycles will be needed for adding the first base of each of the thousands of oligonucleotides being synthesized. Since for addition of one base to each oligomer, four cycles are needed, a maximum of 20 x 4 = 80 cycles will be needed for the production of all possible 20 mers (4²⁰). This method allows the production of very high density (2,50,000 features/cm²) microarrys. However, the synthesis of oligonucleotides longer than 25 mers are difficult with the currently available efficiency of reactions. In future, improvement in photolithographic technology may allow manufacture of microarrays with oligonucleotides containing >25 bases without the need of photomasks.