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Beads for high-throughput nucleic acid analysis

A technology of beads and functional groups, applied in the field of nucleic acid analysis, can solve problems such as difficult sample processing and side reactions

Active Publication Date: 2010-09-22
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, changing the pH has the disadvantage that undesired side reactions can occur and further processing of the sample is more difficult

Method used

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  • Beads for high-throughput nucleic acid analysis
  • Beads for high-throughput nucleic acid analysis
  • Beads for high-throughput nucleic acid analysis

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0163] The preparation of the beads has been disclosed in detail above.

[0164] b) capture nucleic acid molecules of interest from the sample

[0165] The target molecules are then hybridized to beads containing cleavable and non-cleavable primers. Appropriate hybridization conditions in terms of appropriate buffer system and appropriate hybridization temperature are well known in the art, and can be optimized according to specific conditions (such as the length and sequence of specific amplification primers used). Preferably, hybridization is performed using a molar excess of beads compared to the sequence or sequences to be amplified in order to capture as much target nucleic acid as possible. In particular, a molar excess of 1:5 to 1:100, preferably 1:10 to 1:50, has proven to be particularly advantageous. If multiple different target sequences need to be detected and / or analyzed, it is necessary to use a bead library with corresponding multiple different primer pairs. ...

Embodiment 1

[0260] Preparation of primers and photocleavable primers

[0261] Oligonucleotide synthesis was performed on an ABI 394 synthesizer on a 4 x 1 μmol scale. Commercially available tacCPG (Proligo) was used as support material. All other chemicals used in standard synthetic reactions were obtained from Glen Research. Proligo's Phosphoramidite with a tert-butylphenoxy-acetyl protecting group (known as "tac" or "Expedite" monomer) was used. As the capping agent, tert-butylphenoxyacetylacetic anhydride (tac2O) in tetrahydrofuran was used.

[0262] Use the following commercially available modifiers:

[0263] -5' amino modifier C6: (6-(4-one methoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite

[0264] -Spacer phosphoramidite 18 (18-O-dimethoxytritylhexaethylene glycol, 1-[(2-cyanoethyl)-(N,N-diisopropyl)] - Phosphoramidite

[0265] - Photocleavable spacer [4-(4,4'-dimethoxytrityloxy)butyrylaminomethyl)-1-(2-nitrophenyl)-ethyl]-2- Cyanoethyl-(N,N-diisopropy...

Embodiment 2

[0281] Bead preparation and photolysis

[0282] Amino-modified oligonucleotides (Sequence ID #1-9) containing stationary and photocleavable linkers, respectively, were bound to N-hydroxysuccinamide ester (NHS) functionalized agarose beads according to standard methods (Roche / 454-Life Sciences, Branford, CT, USA). chemical reaction mechanism by figure 2 express.

[0283] To trigger photocleavage of the nitrobenzyl linker, the beads were subjected to UV irradiation in a QS1.000 quartz cuvette (1-cm path length) using an 8W dual-wavelength UV lamp (Camag, Berlin, Germany). ) at 366nm. The distance between the quartz cuvette and the UV lamp is 2 cm.

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Abstract

The present invention provides a solid support which is preferably a bead comprising at least two sequence specific amplification primers wherein at least one primer is bound to the support with an inducible cleavable linker. The present invention also provides various method for preparing a solid support comprising at least two sequence specific primers, further characterized in that at least one of the primers is cleavable.

Description

technical field [0001] The invention relates to the field of nucleic acid analysis, specifically miniaturized, highly parallel nucleic acid sequence detection and nucleic acid sequence difference analysis. [0002] The present invention is based on the idea of ​​providing a solid support to which sequence-specific primers are bound, one of which is cleavable and the other is non-cleavable, in order to analyze a specific sequence or to detect SNPs, mutations or any desired The presence of a specific DNA or RNA species of concern. Background technique [0003] Recently, an ultra-high-throughput sequencing system based on pyrosequencing was disclosed, which enables the sequencing of bacterial genomes in substantially no more than a week (WO 04 / 70007; WO 05 / 03375; Margulies, M., et al., Nature 437(2005) 376-80). Starting from sheared genomic DNA, single-molecule fragments are bound to beads that are captured in an emulsion of PCR reaction mixture in oil. Amplification then re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/10
CPCC12Q1/6844C12Q2523/319C12Q2525/197C12Q2563/149C12Q2565/537
Inventor T·弗罗利克D·海因德尔A·罗斯勒
Owner F HOFFMANN LA ROCHE & CO AG
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