Sequential probing of molecular targets based on pseudo-color barcodes with embedded error correction mechanism

A molecular target and barcode technology, which can be used in the determination/inspection of microorganisms, material excitation analysis, fluorescence/phosphorescence, etc., and can solve problems such as limitations, errors, and introduction bias.

Pending Publication Date: 2019-05-24
CALIFORNIA INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Taking nucleic acid detection as an example, current nucleic acid-based assays such as qPCR and microarrays have been useful, but they have not achieved single-molecule sensitivity
On the other hand, next-generation sequencing involves amplification of samples and reverse transcription of mRNA, which may introduce bias and inaccuracy in quantification
Additionally, sample preparation and sequencing can be time-consuming and financially costly
Despite the fact that imaging has been used for the quantification of mRNA transcripts, it is limited to a few hundred genes

Method used

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  • Sequential probing of molecular targets based on pseudo-color barcodes with embedded error correction mechanism
  • Sequential probing of molecular targets based on pseudo-color barcodes with embedded error correction mechanism
  • Sequential probing of molecular targets based on pseudo-color barcodes with embedded error correction mechanism

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Experimental program
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Effect test

Embodiment 1

[0403] Add barcode based on pseudo-color

[0404] Sample Preparation

[0405] Figure 5 Illustrated are mRNA transcripts immobilized on surfaces by poly-A tails or hydrogel embedding. Once cells are lysed, cell lysates or purified total RNA can be immobilized on the surface by coverslips functionalized with DNA or LNAPoly-T by capturing the poly-A tail of the mRNA. Alternatively, mRNA can be mixed with a hydrogel, such as a polyacrylamide gel, and allowed to gel on the surface of a coverslip, where the pores formed will trap mRNA molecules on the surface.

[0406] Primary Probe Design

[0407] Figure 6 An exemplary embodiment of gene-specific primary probe design and sequential barcoding hybridization on mRNA immobilized on a surface is illustrated. in particular, Figure 6 a shows that the primary probe is not directly labeled with a fluorophore. The primary probe comprises (i) a gene-specific targeting region of mRNA, which is 20nt to 35nt, (ii) one or more (mult...

Embodiment 2

[0440] Readout probes and rehybridization in mouse embryonic stem cells

[0441] Synthesis of DNA probe-disulfide-dye conjugates

[0442] Exemplary scheme for the synthesis of disulfide-linked readout probe-dye conjugates. Thiol-modified DNA probes in oxidized form were ordered from Integrated DNA Technologies. Ten nanomolar thiol-modified DNA probes were treated with 10 mM TCEP for 30 minutes at 37°C. After the reduction step and gel column purification, the DNA probe was linked with 50 equivalents of N-hydroxysuccinimide 3-(2-pyridyldithio)propionate (SPDP) in a 1x PBS solution containing 10 mM EDTA. mix in. The mixture was allowed to react at room temperature for at least 2 hours. Immediately after the reaction, the mixture was spin column purified and resuspended in 60uL of 1x PBS containing 100ug of cadaverine dye. The reaction was allowed to proceed at room temperature for at least 4 hours before purification by ethanol precipitation and HPLC. The concentration o...

Embodiment 3

[0449] Brain slice analysis

[0450] As an illustration, barcodes generated using the error correction mechanism disclosed herein were used for in situ transcriptional analysis of single cells, revealing the spatial organization of cells in the mouse hippocampus.

[0451] Identifying the spatial organization of tissues at cellular-level resolution from single-cell gene expression profiles is critical for understanding many biological systems. In particular, there is conflicting evidence as to whether the hippocampus is organized into transcriptionally distinct subregions. Here, a generalizable in situ 3D multiplex imaging approach was applied to quantify hundreds of genes at single-cell resolution by sequentially barcoded fluorescence in situ hybridization (seqFISH) (Lubeck et al., 2014). seqFISH was used to identify unique transcriptional states by quantifying and clustering up to 249 genes in 16,958 cells. By visualizing these clustered cells in situ, we identified distinc...

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Abstract

The present invention, among other things, provides technologies for detecting and/or quantifying nucleic acids in cells, tissues, organs or organisms. Pre-designed barcodes are associated specific molecular targets through sequential hybridization experiments. A pseudo color based barcoding scheme is developed to overcome limitations in the previous generation of the technology such as lack of visual signals that can be associated with the probes or small internal within cell when carrying out in situ experiments.. The current method can be applied to both in vitro and in situ analysis. According to the method, each barcoding round comprises multiple serial hybridizations where a small number of colored signals (that are associated with probes) are used in each hybridization experiment within a serial hybridization round. Images from each serial hybridization experiment within the same serial hybridization round are combined to form a composite image for each barcoding round. In eachbarcoding round, the same set of molecular targets are analyzed. After all barcoding rounds are completed, associated of the barcode with these molecular targets is completed.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to U.S. Patent Application Serial No. 15 / 225,820, filed August 1, 2016, and entitled "Multiplex Labeling of Molecules by Sequential Hybridization Barcoding Using Probes With Cleavable Linkers," filed October 19, 2016 U.S. Patent Application No. 15 / 29,219, filed December 1, 2016, entitled "Error Correction of Multiplex Imaging Analysis by Sequential Hybridization," and entitled "Single Molecule Profiling Through Serial and Barcoded Hybridization" Provisional Application No. 62 / 428,910, U.S. Patent Provisional Application No. 62 / 456,291, filed February 8, 2017, and entitled "Imaging-based Transcriptomic and Translational Profiling of 1000 Genes with in vitro seqFISH," and filed in 2017 US Patent Provisional Application No. 62 / 523,127, filed June 21 and entitled "Transcriptome Profiling of 10,000 mRNAs by RNA SPOTs," each of which is incorporated herein by reference in its entirety. [0003] ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869G01N21/64
CPCC12Q1/6841C12Q1/6813C12Q2525/161C12Q2563/107C12Q2563/179C12Q2565/514C12Q2537/143C12Q2537/149C12Q2565/102C12Q2565/518
Inventor 龙·蔡黄志发希尔·沙哈
Owner CALIFORNIA INST OF TECH
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