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Nucleic Acids Research, Vol 27, Issue 24 4710-4714, Copyright © 1999 by Oxford University Press

ARTICLES

Inversion of in situ synthesized oligonucleotides: improved reagents for hybridization and primer extension in DNA microarrays

M Kwiatkowski, S Fredriksson, A Isaksson, M Nilsson and U Landegren
Rudbeck Laboratory, Department of Genetics, S-75185 Uppsala, Sweden. marek.kwiatkowski@genpat.uu.se

Oligonucleotides synthesized in array format suffer from contamination by truncated species. We have developed a method to invert DNA molecules in situ after completed synthesis. Reactive functions at the 5'-ends of the oligonucleotides are permitted to react with functions on the support before the 3'-ends are released, in effect reversing the orientation of full-length oligonucleotides, while any 5'-truncated molecules are lost. This strategy serves both to purify in situ synthesized reagents and to reorient the oligonucleotides, causing them to expose free 3'-hydroxyls. In situ inverted oligonucleotides can be used in assays based on DNA polymerase-assisted extension of immobilized primers, and we demonstrate their utility in minisequencing and in pyrosequencing.
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