Open AccessInversion of in situ synthesized oligonucleotides: improved reagents for hybridization and primer extension in DNA microarrays
Author(s) -
Marek Kwiatkowski,
Fabrice Simon,
Anders Isaksson,
Mats Nilsson,
Ulf Landegren
Publication year - 1999
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/27.24.4710
Subject(s) - oligonucleotide , biology , in situ , primer extension , dna , microbiology and biotechnology , primer (cosmetics) , dna microarray , combinatorial chemistry , computational biology , biochemistry , base sequence , gene , chemistry , gene expression , organic chemistry
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.