Methods of producing intrachain fluorophore-quencher FRET-aptamers and assays
US7906280B2 · kind B2 · utility
Inventors
Key dates
| Filing date | May 12, 2006 |
| Grant date | Mar 15, 2011 |
| Priority date | — |
| Expiry date | Aug 25, 2026 |
Classification
- Technology area (CPC C)Chemistry; Metallurgy
- CPC primaryC12N2320/10
- WIPO fieldMeasurement
- WIPO sectorInstruments
Abstract
The present invention describes methods for the production and use of single chain (single-stranded) fluorescence resonance energy transfer (“FRET”) DNA or RNA aptamers containing fluorophores (F) and quenchers (Q) at various loci within their structures, such that when its specific matching analyte is bound and the FRET-aptamers are excited by specific wavelengths of light, the fluorescence intensity of the system is modulated (increased or decreased) in proportion to the amount of analyte added. F and Q are covalently linked to nucleotide triphosphates (NTPs), which are incorporated by various nucleic acid polymerases such as Taq polymerase during the polymerase chain reaction (PCR) and then selected by affinity chromatographic, size-exclusion or molecular sieving, and fluorescence techniques. Further separation of related FRET-aptamers can be achieved by ion-pair reverse phase high performance liquid chromatography (HPLC) or other types of chromatography. Finally, FRET-aptamer structures and the specific locations of F and Q within FRET-aptamer structures are determined by digestion with exonucleases and mass spectral nucleotide sequencing analysis.
Source: USPTO / EPO open patent data. Objective bibliographic and citation counts.