RESUMEN
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.
Asunto(s)
ADN/genética , Reacción en Cadena de la Polimerasa/métodos , ARN/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , ADN/química , Sondas de ADN , ADN Bacteriano/química , ADN Bacteriano/genética , Escherichia coli/genética , Humanos , Conformación de Ácido Nucleico , Oligonucleótidos/genética , ARN/química , ARN Bacteriano/química , ARN Bacteriano/genéticaRESUMEN
The syntheses of 1,2-dideoxy-D-ribofuranose and 1,2-dideoxy-1-phenyl-beta-D-ribofuranose are described. Oligodeoxynucleotides containing these analogues have been synthesised and hybridized to their complementary strands. Hypochromicity studies have shown that these duplices are less stable than either the totally complementary duplex or those containing A.C and G.T mismatches.
Asunto(s)
Oligodesoxirribonucleótidos/síntesis química , Oligonucleótidos/síntesis química , Desoxirribosa/análogos & derivados , Desoxirribosa/síntesis química , Código Genético , Hibridación de Ácido Nucleico , Relación Estructura-ActividadRESUMEN
Condensation of N6-benzoyl-2',3'-O-isopropylideneadenosine-5'-aldehyde with nitromethane followed by acid catalyzed acetylation and borohydride reduction leads to N6-benzoyl-9-(5,6-dideoxy-2,3-O-isopropylidene-6-nitro-beta-D-ribo-hexofuranosyl)adenine (4). A second nitroaldol condensation between 4 and N-benzyloxycarbonly-L-aspartic acid-beta-semialdehyde alpha-benzyl ester (5) followed by acetylation and borohydride reduction leads to a fully protected 6'-nitro modification of sinefungin and its C6'-epimer (7). Hydrolysis of the acetonide followed by sequential reduction of the benzyl derived protecting groups and the nitro group and debenzoylation leads to a modest yield of a 3:1 mixture of sinefungin (1) and 6'-episinefungin which can only be separated by analytical ion exchange chromatography.
Asunto(s)
Adenosina/análogos & derivados , Antifúngicos/síntesis química , Adenosina/síntesis química , Cromatografía en Capa Delgada , Indicadores y Reactivos , Isomerismo , Espectroscopía de Resonancia MagnéticaRESUMEN
A solution of benzenesulphonic acid (3%, w/v) in a dimethylformamide and dichloromethane mixture (9:1, v/v) is shown to be a very effective reagent for the detritylation of deoxyoligonucleotides attached to a solid support. The levels of depurination with this reagent were lower than those observed with other reagents such as trichloroacetic acid. Coupling reactions are described using above ambient temperatures with no detectable increase in side products. Both procedures have been successfully incorporated into an automated system, which can compete with the rate of synthesis by the phosphite approach.