Specific detection of DNA and RNA targets using a novel isothermal nucleic acid amplification assay based on the formation of a three-way junction structure
| Autor: | Donald Leonard Nicholas Cardy, Rene Assenberg, Peter Marsh, Philip M. Brown, John S. Lloyd, Susan Deborah Wharam, Anthony Weston, Julie E. McPhee, Trevor Duncan Ray, Graham Andrew Mock |
|---|---|
| Rok vydání: | 2001 |
| Předmět: |
DNA
Bacterial Base pair Oligonucleotides Cystic Fibrosis Transmembrane Conductance Regulator Polymerase Chain Reaction Nucleic acid thermodynamics Escherichia coli Genetics medicine Humans T7 RNA polymerase NAR Methods Online Polymerase biology Oligonucleotide RNA DNA Molecular biology RNA Bacterial Biochemistry RNA spike-in Nucleic acid biology.protein Nucleic Acid Conformation DNA Probes medicine.drug |
| Zdroj: | Nucleic Acids Research. 29:54e-54 |
| ISSN: | 1362-4962 |
| DOI: | 10.1093/nar/29.11.e54 |
| Popis: | The formation of DNA three-way junction (3WJ) structures has been utilised to develop a novel isothermal nucleic acid amplification assay (SMART) for the detection of specific DNA or RNA targets. The assay consists of two oligonucleotide probes that hybridise to a specific target sequence and, only then, to each other forming a 3WJ structure. One probe (template for the RNA signal) contains a non-functional single-stranded T7 RNA polymerase promoter sequence. This promoter sequence is made double-stranded (hence functional) by DNA polymerase, allowing T7 RNA polymerase to generate a target-dependent RNA signal which is measured by an enzyme-linked oligosorbent assay (ELOSA). The sequence of the RNA signal is always the same, regardless of the original target sequence. The SMART assay was successfully tested in model systems with several single-stranded synthetic targets, both DNA and RNA. The assay could also detect specific target sequences in both genomic DNA and total RNA from Escherichia coli. It was also possible to generate signal from E.coli samples without prior extraction of nucleic acid, showing that for some targets, sample purification may not be required. The assay is simple to perform and easily adaptable to different targets. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|