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 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.
Kwiatkowski
M
M
Rudbeck Laboratory, Department of Genetics, S-75185 Uppsala, Sweden. marek.kwiatkowski@genpat.uu.se
Fredriksson
S
S
Isaksson
A
A
Nilsson
M
M
Landegren
U
U
D001483:Base Sequence; D017931:DNA Primers / Q000138:chemical synthesis / Q000737:chemistry*; D007202:Indicators and Reagents; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis* / Q000737:chemistry*; D020411:Oligonucleotide Array Sequence Analysis
D017931:DNA Primers; D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides