Homogeneous Time-Resolved Fluorescence Quenching Assay (TruPoint) for Nucleic Acid Detection
| Autor: | Jari Hovinen, Pia Ollikka, Veli-Matti Mukkala, Annika Elomaa, Harri Hakala, Alice Ylikoski, Ilkka Hemmilä |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
Fluorophore
Nucleic acid quantitation Clinical Biochemistry Analytical chemistry Cystic Fibrosis Transmembrane Conductance Regulator Polymerase Chain Reaction chemistry.chemical_compound Europium Nucleic Acids Complementary DNA Humans Fluorometry Chelation Terbium Chelating Agents Fluorescent Dyes Quenching (fluorescence) Hybridization probe Biochemistry (medical) DNA Fluorescence Combinatorial chemistry chemistry Mutation Spectrophotometry Ultraviolet Time-resolved spectroscopy |
| Zdroj: | Clinical Chemistry. 50:1943-1947 |
| ISSN: | 1530-8561 0009-9147 |
| DOI: | 10.1373/clinchem.2004.036616 |
| Popis: | Laboratories performing genetic screening studies need simplified methods that allow automation for nucleic acid analysis. Ideally, a method should have as few handling steps as possible and allow the use of a closed-tube assay format to reduce the risk of PCR contamination. Different homogeneous assay chemistries have been exploited to integrate amplification and detection to allow simple nucleic acid analysis with minimal post-PCR handling (1)(2)(3)(4)(5). Double-stranded DNA probes consisting of a 5′-terminus-labeled fluorescent strand and a complementary strand labeled on the 3′ terminus with a quencher have been exploited in homogeneous hybridization (2)(6)(7). Fluorescence signal is generated when the fluorophore strand hybridizes with the target. When the quencher strand hybridizes with the fluorophore strand, the signal from the fluorophore is quenched. Morrison et al. (6) used probes consisting of strands of equal length and a competitive hybridization technology, whereas others have exploited probes consisting of strands with different lengths for detection of PCR products (2)(7). The use of stable and fluorescent lanthanide chelates has enabled the development of homogeneous assay technologies based on time resolution (8). Time-resolved fluorescence-quenching assays (TR-FQAs), based on energy transfer from a lanthanide chelate to a nonfluorescent quencher, have been applied in various assays of hydrolyzing enzymes (9)(10) and are commercialized as TruPoint™. The first homogeneous PCR assays with lanthanide-labeled probes used an environmentally sensitive terbium chelate, the fluorescence intensity of which changes on probe hybridization (3)(11). Subsequently, the technology was further improved by use of additional quencher probes (12). Recently, a combination of quenching and competitive hybridization has been applied for end-point detection of PCR products (13). The aim of the present study was to compare two homogeneous PCR assay approaches based on a … |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|