In situ enzymatic template replication on DNA microarrays.

Abstract

DNA microarrays are very useful tools to study the realm of nucleic acids interactions at high throughput. The conventional approach to microarray synthesis employs phosphoramidite chemistry and yields unmodified DNA generally attached to a surface at the 3' terminus. Having a freely accessible 3'-OH instead of 5'-OH is desirable too, and being able to introduce nucleoside analogs in a combinatorial manner is highly relevant in the context of nucleic acid therapeutics and in aptamer research. Here, we describe an enzymatic approach to the synthesis of high-density DNA microarrays that can also contain chemical modifications. The method uses a standard DNA microarray, to which a DNA primer is covalently bound through photocrosslinking. The extension of the primer with a DNA polymerase yields double-stranded DNA but is also amenable to the incorporation of modified dNTPs. Further processing with T7 exonuclease, which catalyzes the degradation of DNA in a specific (5'→3') direction, results in template strand removal. Overall, the method produces surface-bound natural and non-natural DNA oligonucleotides, is applicable to commercial microarrays and paves the way for the preparation of combinatorial, chemically modified aptamer libraries.