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Multiplex analysis of target nucleic acids

A target nucleic acid and particle technology, applied in the field of multiple analysis of target nucleic acid, can solve the problems of low sensitivity, cross-reactivity or analysis/instrument complexity of non-specific targets

Active Publication Date: 2021-02-02
FIREFLY BIOWORKS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, many of these methods are limited by lower sensitivity, cross-reactivity, or analytical / instrumental complexity for non-specific targets

Method used

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  • Multiplex analysis of target nucleic acids
  • Multiplex analysis of target nucleic acids
  • Multiplex analysis of target nucleic acids

Examples

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example 1

[0209] Example 1: MicroRNA Capture and Signal Amplification

[0210] This example demonstrates that exemplary particles can capture microRNAs and amplify their signal via PCR or PCR-like reactions and generation of fluorescent products. Exemplary methods are described in detail below.

[0211] MicroRNAs are an emerging class of small RNA biomarkers that have been shown to regulate most genes in eukaryotic organisms. These RNA molecules also showed higher stability in blood. Several emerging technologies have managed to accurately detect and quantify microRNAs in multiplexed formats, but many have poor sensitivity, low multiplex, or low sample yields. The methods outlined above are well suited for ultrasensitive detection of these and other small RNA markers. In this embodiment, the solid or semi-solid support will comprise a mixture of one or more types of hydrogel particles each carrying a unique code and capture probe. MicroRNA targets will be captured and engaged via li...

example 2

[0244] Example 2. mRNA capture and signal amplification

[0245] This example demonstrates that exemplary particles can capture mRNA and amplify its signal via PCR or PCR-like reactions and generation of fluorescent products. Exemplary methods are described below.

[0246] mRNA molecules are key protein-coding transcripts. These RNAs are typically on the order of kilobases in length and are present in relatively low copy numbers compared to other transcript molecules. Highly sensitive multiplex methods are required to measure the expression of these gene transcripts due to their low copy numbers and high degradation rates, and the fact that humans alone have over 20,000 protein-coding mRNAs. The methods described above can be modified to capture and measure these and other longer transcripts. Likewise, multiple solid or semi-solid supports each carrying a unique capture probe can be utilized to capture the respective mRNA targets of interest. A ligation reaction will be us...

example 3

[0250] Example 3. lncRNA detection and signal amplification

[0251] This example demonstrates that exemplary particles can capture lncRNAs and can amplify their signal via PCR or PCR-like reactions and generation of fluorescent products. Exemplary methods are described below.

[0252] Additional longer non-coding RNA transcripts including lincRNAs are increasingly of interest to life science researchers and clinical molecular researchers. The methods described above for microRNA and other small RNA targets can be adapted for longer RNA targets. These targets will require alternative probe designs because synthetic oligonucleotides larger than 100 nucleotides quickly become complex and are expensive to manufacture. Such as Figure 10 As demonstrated in , it is possible to attach universal oligonucleotide adapters to each end of longer transcripts by capturing the 3' and 5' ends of targeted longer RNAs and enabling many RNA targets to adopt circular or globular conformations...

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Abstract

The present invention provides, inter alia, a method of detecting a target nucleic acid comprising the steps of: a) bringing a sample together with one or more capture probes each comprising at least one target capture sequence while allowing said one or more capture probes to capture said sample b) amplifying the captured one or more target nucleic acids in a reaction mixture comprising a detectable entity such that the amplified one or more target nucleic acids are labeled with the A detectable entity; c) incubating the amplification product with a plurality of recapture probes such that the amplified one or more target nucleic acids are recaptured by the plurality of recapture probes; and d) detecting the resultant and recapture A signal produced by a detectable entity bound to the amplified one or more target nucleic acids, wherein the presence and / or abundance of the detectable signal indicates the presence and / or presence of the one or more target nucleic acids in the sample or abundance.

Description

[0001] related application [0002] This application claims priority and benefit to U.S. Provisional Patent Application Nos. 61 / 816,070, filed April 25, 2013, and 61 / 936,826, filed February 6, 2014, the entire contents of which are incorporated by reference incorporated into this article. [0003] sequence listing [0004] Pursuant to 37 CFR 1.52(e)(5), the Sequence Listing as a text file (titled "SequenceListing.txt", created on April 25, 2014, and 5 kilobytes in size) is incorporated by reference in its entirety In this article. Background technique [0005] Nucleic acids are becoming increasingly important for both diagnostic and therapeutic uses. For example, early and precise detection of various nucleic acid biomarkers or genomic mutations and imbalances present in diseased cells may have important clinical implications. Multiplexing is a powerful tool for analyzing nucleic acids, especially in laboratory and diagnostic settings. However, many of these methods ar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6806C12Q1/682
CPCC12Q1/682C12Q2525/155C12Q2525/191C12Q2525/207C12Q2563/149C12Q2563/179C12Q1/6806
Inventor 达尼埃尔·C·普赖格伊邦伊萨克·斯托纳安东尼·富斯科迈克尔·R·塔克特
Owner FIREFLY BIOWORKS