The Synthesis of Branched Oligonucleotides as Signal Amplification Multimers for Use in Nucleic Acid Assays

Horn, Thomas; Warner, Brian D.; Running, Joyce A.; Downing, Kristi; Clyne, Jennifer; Urdea, Mickey S.

doi:10.1080/07328318908054234

Cited by 8 publications

(6 citation statements)

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“…We have evaluated different synthetic probes, including branched probes (Horn et al 1989), padlock probes (Nilsson et al 1994), and conventional single-stranded oligodeoxynucleotide probes at various lengths with respect to their hybridization efficiencies. Different types of labels in different positions have been tested, together with different detection systems.…”

Section: Design Of Probesmentioning

confidence: 99%

Non-radioactive in situ hybridization for mRNA with emphasis on the use of oligodeoxynucleotide probes

Hougaard

Hansen

Larsson

1997

Histochemistry and Cell Biology

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Section: Design Of Probesmentioning

confidence: 99%

Non-radioactive in situ hybridization for mRNA with emphasis on the use of oligodeoxynucleotide probes

Hougaard

Hansen

Larsson

1997

Histochemistry and Cell Biology

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“…Several signal amplification strategies have been proposed, either based on the introduction of multiple reporter groups such as biotin (2), fluorophores (3), or 2,4-dinitrophenyl (4) in the ODN used for the detection or focusing on the use of branched oligonucleotides (5,6) in combination with detection probes labeled with an enzyme. However, the increase of sensitivity obtained by these routes is still limited.…”

Section: Introductionmentioning

confidence: 99%

Water-Soluble Dendrigrafts Bearing Saccharidic Moieties: Elaboration and Application to Enzyme Linked OligoSorbent Assay (ELOSA) Diagnostic Tests

et al. 2005

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The synthesis of a series of water-soluble galactopyranose-functionalized polystyrene-polyvinyl ether dendrigrafts and their characterization (in solution and thin solid deposits) have been achieved. The presence of external galactopyranose groups on dendritic polymers has been exploited to prepare dendrigraft-oligonucleotide conjugates using a simple one-step coupling procedure with amino-ended oligonucleotides (ODNs). Several parameters such as the peripherical density of hydrophilic branches, the polymerization degree of polystyrene or poly(hydroxyethyl vinyl ether) blocks, and the number of galactopyranose groups were tuned. A capture test with short labeled complementary ODNs (25 bases) confirmed the presence of covalently bound ODNs on various kinds of dendrigrafts. The ability of the dendritic polymers to enhance the sensitivity of enzyme-linked oligosorbent assay (ELOSA) diagnostic tests (detection of hepatitis B virus, DNA target of 2400 bases) was then evaluated, especially the influence of the macromolecular architecture and the impact of the structural parameters. The dendrigraft-ODN conjugate with the lower saccharide external density was found to lead to a very significant amplification of the fluorescence signal, corresponding to a limit of sensitivity of 10(9) DNA copies per milliliter (instead of 10(11) DNA copies per milliliter without using dendrigrafts). Conversely, the dendrigrafts exhibiting a very high number of branches and galactopyranose groups at their periphery were not able to induce a better sensitivity due to steric hindrance generated by the peripheral congestion on these polymers.

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“…Such amplification may be particularly important in in situ hybridisation and in the emerging techniques which exploit oligonucleotide arrays (22)(23)(24), where the signal is limited by the surface density of the oligonucleotides or the target molecule. Phosphitamide reagents have been described which double the amount of reactive 5′-hydroxyl groups after each condensation step thus giving 2 n reactive OH-groups after n condensations (25)(26)(27)(28)(29)(30); they have been used in combination with biotin, fluorescein, pyrene and other phosphitamide synthons for multiple 5′-labelling of oligonucleotides. But all these compounds share a difficulty: too dense a concentration of reporter groups leads to self-quenching of fluorescence.…”

Section: Introductionmentioning

confidence: 99%

Oligonucleotide dendrimers: synthesis and use as polylabelled DNA probes

Shchepinov

Udalova

Bridgman

et al. 1997

Nucleic Acids Research

141

106

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Oligonucleotide dendrimers were synthesized using a novel phosphoramidite synthon, tris-2,2,2-[3-(4,4'-dimethoxytrityloxy) propyloxymethyl]ethyl- N , N -diisopropylaminocyanethoxy phosphoramidite. Label, incorporated using [gamma-32P]ATP and polynucleotide kinase, was increased in proportion to the number of 5'-ends. There was a similar increase in signal when these multiply labelled oligonucleotides were used as probes to oligonucleotide arrays. A dendrimeric oligonucleotide was used successfully as a primer in the PCR. The strand bearing the dendrimer was resistant to degradation by T7 Gene 6 exonuclease making it easy to convert the double-stranded product of the PCR to a multiply-labelled, single-stranded probe.

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The Synthesis of Branched Oligonucleotides as Signal Amplification Multimers for Use in Nucleic Acid Assays

Cited by 8 publications

References 0 publications

Non-radioactive in situ hybridization for mRNA with emphasis on the use of oligodeoxynucleotide probes

Non-radioactive in situ hybridization for mRNA with emphasis on the use of oligodeoxynucleotide probes

Water-Soluble Dendrigrafts Bearing Saccharidic Moieties: Elaboration and Application to Enzyme Linked OligoSorbent Assay (ELOSA) Diagnostic Tests

Oligonucleotide dendrimers: synthesis and use as polylabelled DNA probes

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