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Arrays for single molecule detection and uses thereof

An array and sequence technology, applied in the field of manufacturing and using spatially addressable low-density or high-density molecular arrays, which can solve the problems of complex samples causing difficulty, separation, and difficulty in resolving heterogeneous events.

Inactive Publication Date: 2019-03-15
SINGULAR BIO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] 2. Rare events in the background of common events at a particular locus are impossible to detect body-wise because they are masked by more common events
[0010] 3. Difficulty parsing heterogeneous events
For example, it is difficult to separate the contribution (or lack thereof) from false signals such as retracement, mispriming, or self-priming from true signals based on measured interactions
[0011] 4. Difficulties caused by complex samples such as genomic DNA or mRNA populations
[0012] 5. The holistic nature of conventional methods does not allow access to specific features of individual molecules (especially more than one feature)
Ensemble analysis fails to resolve haplotypes from heterozygous samples
Currently available molecular biology techniques, such as allele-specific or single-molecule PCR, are difficult to optimize and scale up
[0013] 6. Transient process is difficult to analyze
However, the cost of large-scale resequencing by this method remains high, with only 65% ​​of probed bases giving results with sufficient base calling confidence

Method used

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  • Arrays for single molecule detection and uses thereof
  • Arrays for single molecule detection and uses thereof
  • Arrays for single molecule detection and uses thereof

Examples

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preparation example Construction

[0369] The present invention involves combining the preparation of single molecule arrays with performing assays on the single molecule arrays. Especially when either or both are combined with detection / imaging of individual probes on a substrate as described herein and assays based on counting single molecules or recording and measuring signals from single molecules.

[0370] The invention also provides software and algorithms for processing data from the methods described above.

[0371] A system for detecting genetic variation according to the methods described herein includes a variety of elements. Some elements include converting a raw biological sample into a useful analyte. The analyte is then detected and data generated which is later processed into a report. Figure 19 Various modules that can be included in the system are shown in . Figure 20 More details of the various methods used to analyze the data (eg including image processing) are shown in . Analysis can ...

Embodiment approach

[0753] Figure 21 is an implementation of the assay used to quantify genomic copy number at two genomic loci. In this embodiment of the assay, 105 and 106 are target molecules. 105 contains the sequence corresponding to the first genomic locus "locus 1" corresponding to the query copy number (e.g., chromosome 21), and 106 contains the sequence corresponding to the second genomic locus "locus 2" corresponding to the query copy number (e.g., chromosome 18). sequence. Figure 21 An example of one probe set per genomic locus is included, but in some embodiments of the assay, multiple probe sets can be designed to interrogate multiple regions within a genomic locus. For example, greater than 10, greater than 100, or greater than 500 probe sets corresponding to chromosome 21 can be designed. Figure 21 Only a single probe set for each genomic locus is illustrated, but importantly the scope of the invention allows the use of multiple probe sets for each genomic locus. Figure 21 ...

Embodiment 1

[0911] Example 1 - Cleaning the Substrate

[0912] The following steps are preferably performed in a clean room. Thoroughly clean pure white glass plates / slides (Knittel Glazer, Germany) (which can be polished for flatness) or Spectrosil slides by, for example, ultrasonication in a surfactant solution (2% Micro-90) for 25 minutes. surface, washed in deionized water, rinsed thoroughly with milliQ water, and immersed in 6:4:1 milliQ H 2 O:30%NH 4 OH:30%H 2 o 2 or immersed in H 2 SO 4 / CrO 3 1.5 hours in the cleaning solution. After cleaning, the plates are rinsed and stored in a dust-free environment, for example under milliQ water. The top layer of the mica substrate was peeled off by covering with scotch tape and quickly tearing off the layer.

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Abstract

The invention relates to methods of detecting a genetic variation in a genetic sample from a subject using labeled probes and counting the number of labels in the probes. The invention also relates tomanufacturing and using arrays and analytical approaches based on single molecule detection techniques.

Description

Background technique [0001] The present invention relates to methods of detecting genetic variation in a genetic sample from a subject. Detecting genetic variation is important in many aspects of human biology. The invention also relates to the fabrication and use of spatially addressable low or high density molecular arrays and assays based on single molecule detection techniques. [0002] Advances in the Human Genome Project have created the need to: (i) analyze the expression characteristics of genes and gene products, and (ii) analyze the variation of genes and genomes. This has led to great interest in massively parallel research methods. The success of using DNA markers in finding genes for monogenic disorders and the recent proposal for large-scale association studies to dissect complex traits has further fueled interest in developing new methods for detecting variants. Large-scale studies and high-throughput screening are also required in the pharmaceutical industry...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12Q1/6827C12Q1/6834C40B30/04G01N21/64G01N33/52
CPCC12Q1/6827C12Q1/6837B01J19/0046B01J2219/00529B01J2219/00576B01J2219/00596B01J2219/00608B01J2219/00621B01J2219/00659B01J2219/00677B01J2219/00702B01J2219/00722C12Q2563/107C12Q2565/507C12Q2565/519C12N15/1065C12Q1/68
Inventor P·J·柯林斯H·B·琼斯
Owner SINGULAR BIO
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