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A droplet-based method and apparatus for composite single-cell nucleic acid analysis

A cell and droplet technology, applied in biochemical equipment and methods, recording carriers used by machines, DNA preparation, etc., can solve the problem of high cost

Active Publication Date: 2018-04-03
THE BROAD INST INC +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Conducting studies that require data interpretation at the single-cell (or single-molecule) level can be challenging or cost-prohibitive at best
While techniques or instruments exist for single-molecule or single-cell analysis (e.g., digital polymerase chain reaction (PCR) or Fluidigm C1, respectively), none currently allow for the dynamic delivery of reagents and / or Scalable method of attaching molecular "information" to individual reactions so that large numbers of reactions / assays can be jointly processed and analyzed while still maintaining the ability to partition results by individual reactions / assays

Method used

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  • A droplet-based method and apparatus for composite single-cell nucleic acid analysis
  • A droplet-based method and apparatus for composite single-cell nucleic acid analysis
  • A droplet-based method and apparatus for composite single-cell nucleic acid analysis

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Embodiment 1

[0162] In this protocol, uniquely barcoded beads were synthesized for use as primers for reverse transcription. Beads are first prepared with immobilized sequences synthesized on the surface ( Figure 2A A) starts with the SMT in A), which serves as a priming site for downstream PCR. Next, the beads were split and pooled a total of 12 times into four identical reaction vessels to generate 4^12 unique barcode sequences unique to each bead ( Figure 2B ). This 12bp region was used as the cell barcode as it was specific for each bead. Next, the beads are all pooled together for 8 rounds of degenerate synthesis using all four bases; this 8bp region is the "molecular barcode" and will uniquely tag each mRNA so that each mRNA molecule in the cell can Count digitally. Finally, the 30dT base is synthesized, which serves as a capture region for the polyadenylation tail of the mRNA (often referred to as "oligo-dT" in the literature).

[0163] Synthesis of Uniquely Barcoded Beads

...

Embodiment 2

[0190] Example 2: Genome-wide expression profiling of thousands of individual cells using nanoliter microdroplets

[0191] Disease occurs in complex tissues made of different types of cells and (almost) never involves a single cell acting on itself: cells always interact with each other to make collective decisions, collaborate dynamics, and function together. In normal tissues, this leads to homeostasis; in disease, dysfunction of one or more interacting functions can lead to or exacerbate a pathological state.

[0192] Cells, the fundamental units of biological structure and function, vary widely in type and state. Single-cell genomics can characterize cellular identity and function, but limitations of simplicity and scale have prevented its widespread application. Applicants here describe Drop-Seq, which is a rapid method of analyzing the RNA of each cell by isolating thousands of individual cells into nanoliter-sized aqueous droplets, barcoding the RNA of each cell, and s...

Embodiment 3

[0265] Embodiment 3: the extended experiment process of embodiment 2

[0266] Bead synthesis. Bead functionalization and reverse phosphoramidite synthesis (5'-3') were performed by Chemgenes Corp. Toyopearl HW-65S resin (average particle size of 30 microns) was purchased from Tosoh Biosciences, and the surface alcohols were functionalized with PEG derivatives to create 18-carbon long flexible chain linkers. Functionalized beads were then used as solid supports for reverse phosphoramidite synthesis (5'→3') of DNA synthesis on an Expedite 8909 DNA / RNA synthesizer using a 10 μmol cycle scale and a coupling time of 3 minutes. The amides used were: N 6 -Benzoyl-3'-O-DMT-2'-deoxyadenosine-5'-cyanoethyl-N,N-diisopropyl-phosphoramidite (dA-N 6 -Bz-CEP);N 4 -Acetyl-3'-O-DMT-2'-deoxy-cytidine-5'-cyanoethyl-N,N-diisopropyl-phosphoramidite (dC-N 4 -Ac-CEP); N 2 -DMF-3′-O-DMT-2′-deoxyguanosine-5′-cyanoethyl-N, N-diisopropyl-phosphoramidite (dG-N 2 -DMF-CEP); and 3'-O-DMT-2'-deoxythy...

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Abstract

The present invention generally relates to a combination of molecular barcoding and emulsion-based microfluidics to isolate, lyse, barcode, and prepare nucleic acids from individual cells in a high-throughput manner.

Description

[0001] Related Applications and Incorporation by Introduction [0002] This application claims the benefit and priority of U.S. Provisional Patent Application Serial No. 62 / 048,227, filed September 9, 2014, and U.S. Provisional Patent Application Serial No. 62 / 146,642, filed April 13, 2015. [0003] The foregoing application and all documents cited therein or during the prosecution thereof (“Application Citations”) and all documents cited or referenced in said Application Citations, and all documents cited or referenced herein (“herein Documents cited") and all documents cited or referenced in said documents cited herein, together with any manufacturer's instructions, descriptions, product brochures, and The Product Insert together is hereby incorporated herein and may be used in the practice of the present invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually ind...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68G06K19/06
CPCC12N15/1096C12Q1/6809C12Q1/6834C12Q1/6869G06K19/06C12Q2521/107C12Q2563/159C12Q2565/629C12Q2525/173C12Q2563/149C12Q2525/155C12Q2525/15C12Q2525/161C12Q2563/179
Inventor A·雷格夫E·Z·麦科斯科S·A·麦卡罗尔A·K·沙勒克A·巴苏C·B·福特H·帕克D·A·韦茨
Owner THE BROAD INST INC
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