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Multiplexed solid-phase nucleic acid amplification assay

A nucleic acid and target nucleic acid technology, applied in the field of nucleic acid detection

Inactive Publication Date: 2007-04-18
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Prior to the present invention, immobilized dual-labeled oligonucleotide probes carrying a label moiety that can be released by 5'-nuclease activity had not been used in nucleic acid amplification reactions, or had not been used in nucleic acid amplification reactions. real-time monitoring of reaction

Method used

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Embodiment 1

[0134] Probe and Primer Synthesis

[0135]Using standard phosphoramidite chemistry (S.L Beucage, Methods in Molecular Biology, Vol. 20: Protocols for Oligonucleotides and Analogs, pp. 33-61), oligonucleotides were prepared on glass with controlled wells using an Applied Biosystems 394 DNA synthesizer. nucleotide primers. Phosphoramidite analogs of deoxyadenosine, deoxycytidine, deoxyguanosine, and thymidine were purchased from Applied Biosystems (Foster City, California) and diluted to 0.1 M in anhydrous acetonitrile before use. Coupling, oxidation and deprotection reagents were also purchased from Applied Biosystems and used according to the manufacturer's recommendations. t-Butylbenzyl-dA CPG was prepared as previously described (see US Patent No. 6,001,611). Oligonucleotide probes were prepared using dye-phosphoramidites from BioGenex (San Ramon, California) and the 3'-amino modifier C7CPG (C7NH2) from Glen Research (Sterling, Virginia).

[0136] The following modified o...

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Abstract

The present invention provides a method for detecting the amplification of a target nucleic acid using an oligonucleotide probe immobilized on a solid substrate through its 3' end. The invention further relates to devices, reaction mixtures and kits for carrying out the method.

Description

technical field [0001] The invention belongs to the field of nucleic acid detection. Background technique [0002] Detection of amplification of nucleic acid sequences using liquid-phase double-labeled oligonucleotide probes in combination with an enzyme having 5' nuclease activity is well known in the art (see, e.g., U.S. Pat. Nos. 5,210,015; 5,487,972; 5,804,375; and Holland, et al., Proc. Natl. Acad. Sci USA (1988) 88:7276-80). Prior to the present invention, immobilized dual-labeled oligonucleotide probes carrying a label moiety that can be released by 5'-nuclease activity had not been used in nucleic acid amplification reactions, or had not been used in nucleic acid amplification reactions. Real-time monitoring of the reaction. [0003] There remains a need for more efficient and convenient methods for simultaneously detecting the amplification of multiple nucleic acid sequences. The present invention fulfills this need and others. Contents of the invention [000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6823C12Q1/6837
Inventor D·波拉特S·G·威尔
Owner F HOFFMANN LA ROCHE & CO AG