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

Oligonucleotides synthesized in array format suffer from contamination by t runcated species. We have developed a method to invert DMA molecules in sit u after completed synthesis. Reactive functions at the 5'-ends of the oligo nucleotides are permitted to react with functions on the support before the 3'-ends are released, in effect reversing the orientation of full-length o ligonucleotides, while any 5'-truncated molecules are lost. This strategy s erves both to purify in situ synthesized reagents and to reorient the oligo nucleotides, causing them to expose free 3'-hydroxyls. In situ inverted oli gonucleotides can be used in assays based on DNA polymerase-assisted extens ion of immobilized primers, and we demonstrate their utility in minisequenc ing and in pyrosequencing.