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Miniaturized, high-throughput nucleic acid analysis

a nucleic acid analysis and high-throughput technology, applied in the field of nucleic acid analysis, can solve the problem of difficult sample processing and achieve the effect of difficult sample processing

Inactive Publication Date: 2010-09-30
ROCHE MOLECULAR SYST INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0023]KR 2007044677 describes beads with a first and a second immobilized PCR primer for use in emulsion PCR. The first primer is non cleavable. The release of the second primer is achiev

Problems solved by technology

Yet, changing the pH value has the disadvantage of potentially undesired side reactions and furthermore makes further processing of the sample more difficult.

Method used

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  • Miniaturized, high-throughput nucleic acid analysis

Examples

Experimental program
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example 1

Preparation of Primers and Photocleavable Primers

[0194]Oligonucleotide synthesis was carried out on a 4 times 1 μmol scale on an ABI 394 synthesizer. Commercially available tac CPG (Proligo) was used as the support material. All other chemicals for the standard synthesis were obtained from Glen Research. Phosphoramidites with tert. butylphenoxy-acetyl protective groups (known as “tac” or “Expedite” monomers) from Proligo were used. As capping reagent tertbutylphenoxyacetyl acetic anhydride (tac2O) in tetrahydrofuran was used.

[0195]The following commercially available modifiers were used:[0196]5′ amino modifier C6: (6-(4-Monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite[0197]Spacer phosphoramidite 18 (18-O-Dimethoxytritylhexaethyleneglycol, 1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite[0198]Photocleavble spacer [4-(4,4′-Dimethoxytrityloxy)butyramidomethyl)-1-(2-nitrophenyl)-ethyl]-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidite[0199]BiotindT phosphor...

example 2

Preparation of Beads and Photocleavage

[0205]Amino-modified oligonucleotides containing stationary and photo-cleavable linker (sequence-ID #1-9) respectively, were conjugated to N-hydroxysuccinimide ester (NHS) functionalized sepharose beads (Roche / 454-Life Sciences, Branford, Conn., USA) according to the standard protocol. The chemical reaction mechanism is represented in FIG. 2.

[0206]To trigger photocleavage of the Nitrobenzyl linker these beads were subjected to UV irradiation in a QS1.000 quartz cell (1-cm path length) using a 8 W dual wavelength UV lamp (Camag, Berlin, Germany) at 366 nm. The distance between quartz cell and UV lamp was 2 cm.

example 3

Analysis of the Photocleavage Reaction (Corresponding to FIG. 3)

[0207]This example describes the detection of the photocleavage of fluorescein-modified oligonucleotide probes immobilized to sepharose beads.

[0208]5×106 of beads conjugated with fluorescein-modified oligonucleotide probes (sequence-ID #1) were extensively washed, then suspended in 100 μl 50 mM Tris / HCl pH 7.5 buffer and irradiated for 15 min as described in example 2. Subsequently the beads were centrifuged and the supernatant was analyzed for photocleaved fluorescein-modified oligonucleotides (FAM) by absorbance measurement using a Nanoprop 1000 spectrophotometer (Thermo Scientific, Waltham, Mass., USA) (FIG. 3A).

[0209]In addition, 0.6×106 beads carrying fluorescein-modified oligonucleotide probes (sequence-ID #1) were suspended in 800 μl PCR-reaction mixture (Table 3).

TABLE 3ReagentFinal concentrationTween-800.01%BSA0.10%MgSO42.5mMGlycerol 3.6%Tris-H2SO4 pH 8.958.1mMNH4—SO417.4mMdATP, dGTP, dCTP, dTTPeach 0.40mMExpan...

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Abstract

The present invention is directed to method for analyzing multiple nucleic acid molecules of interest comprising in the steps of (i) providing a plurality of beads, characterized in that each bead comprises at least two sequence specific amplification primers, further characterized in that at least one of the primers is bound to the bead via a cleavable linker, (ii) capturing the nucleic acid molecules of interest from a sample, (iii) clonally isolating the plurality of beads, (iv) cleaving the at least one primer, (v) clonally amplifying the nucleic acid thereby creating multiple amplification products, and (vi) analyzing the amplification products.

Description

[0001]The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 20, 2010, is named 25938US.txt and is 2,777 bytes in size.RELATED APPLICATIONS[0002]This application claims priority to European application EP 09002627.9 filed Feb. 25, 2009.FIELD OF THE INVENTION[0003]This invention relates to the area of nucleic acid analysis and in particular to the miniaturized, highly parallel detection of nucleic acid sequences and analysis of differences in nucleic acid sequences.[0004]The invention is based on the idea of providing a solid support to which sequence specific primers are bound, with one being cleavable and the other being non-cleavable, in order to analyze specific sequences or detect the presence of a SNP, a mutation or any particular DNA or RNA species of interest.BACKGROUND OF THE INVENTION[0005]Recently, an ultra-high throughput sequencing system based on p...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6834C12Q1/6846C12Q1/6874C12Q2563/149C12Q2525/197C12Q2523/319C12Q2565/537C12Q2547/101C12Q2537/143
Inventor FROEHLICH, THOMASHEINDL, DIETERROESLER, ANGELIKA
Owner ROCHE MOLECULAR SYST INC
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