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Methods and means for nucleic acid sequencing

A nucleic acid sequencing and nucleic acid technology, applied in the field of determining the one or more reference sequences, possibly one or high-throughput sequencing, the sequencing disclosed in PCT/EP2005/002870, using reference sequences related to templates, can Solve the problems of huge sequencing burden and large CR to achieve effective analysis and identification, and cost-effective analysis and identification

Inactive Publication Date: 2009-06-17
GENIZON BIOSCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, CRs can be quite large (>100kb), so there is a huge sequencing burden to sequence many CRs in many individuals

Method used

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  • Methods and means for nucleic acid sequencing
  • Methods and means for nucleic acid sequencing
  • Methods and means for nucleic acid sequencing

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preparation example Construction

[0143] The present invention advantageously uses rolling circle amplification to synthesize random arrays from a sample containing multiple template molecules in a single reaction. can be achieved up to 10 5 -10 7 per mm 2 Density. The random array synthesis method used in the embodiments of the present invention may include:

[0144] a. Provide a surface (eg glass) with an activated surface.

[0145] b. Attachment of primers, preferably via covalent bonds, or instead of covalent bonds, strong non-covalent bonds (eg biotin / streptavidin) can be used.

[0146] b. Addition of circular single-stranded template, preferably at a density suitable for the detection device.

[0147] c. Anneal the template to the primer.

[0148] d. Amplification using rolling circle amplification to generate long single-stranded tandem repeat templates that are attached to the surface at various locations.

[0149] (See eg Lizardi et al., for a description of "Mutation detection and single-molec...

Embodiment 1

[0237] PREPARATION OF DNA TEMPLATES FOR CANTALOUPE

[0238] enter

[0239] Double-stranded DNA template.

[0240] Template break:

[0241] Using the restriction enzyme CviJ I * (EURx, Poland), an enzyme that recognizes 5'-GC-3' and cuts blunt ends between them. Prepare the restriction reaction as follows:

[0242] 1 μg template 1.5 μg template 2 μg template 2x reaction buffer

25μl 2x reaction buffer

25μl 2x reaction buffer

25μl 0.3 units of CviJ I * 0.3 units of CviJ I * 0.3 units of CviJ I * Add water to 50μl Add water to 50μl Add water to 50μl total capacity

50μl total capacity

50μl total capacity

50μl

[0243] The reaction was incubated at 37°C for 1 hour.

[0244] The cleaved DNA was purified using a PCR cleanup kit (Qiagen) according to the manufacturer's protocol.

[0245] A fraction was analyzed on a 2% agarose gel to determine optimal reaction conditions for a particular batch of te...

Embodiment 2

[0281] Preparation of candidate region-enriched fragments for CANTALOUPE sequencing technology

[0282] Step 1: Selection of regions for enrichment and probe preparation

[0283] To enrich candidate regions of interest from nucleic acid samples, the following exemplary methods can be used prior to sequencing using Cantaloupe technology.

[0284] Based on genome-wide association studies of diseases or complex genetic traits such as Crohn's disease and psoriasis, the average candidate region size is approximately 500,000 bases (0.5 Mb). All candidate regions associated with the disease could be selected, but in this example, 3 different regions from different chromosomes were selected (H region: 453.5kb, R region: 285.5kb and E region: 193.6kb), which covered a total of 932.6 kb. Furthermore, in a separate example, only the E region (193.6 kb) was selected to verify the effect of size on the enrichment method of the present invention.

[0285] The probe sets used in this me...

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Abstract

The present invention provides a nucleic acid sequencing method. The method comprises enriching a nucleic acid sample for target nucleic acids, where the nucleic acid sample is enriched through at least a first round of hybridization selection and amplification, and a second round of hybridization selection and amplification. The enriched nucleic acids are in a form convenient for sequencing with the Cantaloupe sequencing technology, which employs shotgun sequencing by hybridization (SBH) of immobilized rolling circle amplicons.

Description

[0001] This application claims the benefit of US Provisional Application 60 / 781,731, filed March 14, 2006, the entire contents of which are hereby incorporated by reference. [0002] The present invention relates to nucleic acid sequencing, in particular to the sequencing method disclosed in PCT / EP2005 / 002870 (corresponding to WO2005 / 093094), the entire contents of which are incorporated herein by reference. Background of the invention [0003] Although many different approaches are used in genome research, direct sequencing is by far the most important. In fact, if sequencing can be made efficient, the 3 main levels of genomic analysis (sequence determination, genotyping and gene expression analysis) can be elucidated. For example, model species can be sequenced, individuals can be genotyped by whole genome sequencing, and RNA populations can be thoroughly analyzed after conversion to cDNA. [0004] Other assays that could be improved by advances in sequencing technology inc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12P19/34C12M1/36
CPCC12Q1/6874C12Q1/6806C12Q1/6855
Inventor A·贝路驰S·若弗鲁瓦S·林纳尔松P·贝鲁布T·基思
Owner GENIZON BIOSCI
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