IP Verse

Barcoded Beads And Method For Making The Same By Split-Pool Synthesis

Patent No. US11692214 (titled "Barcoded Beads And Method For Making The Same By Split-Pool Synthesis") was filed by Roche Sequencing Solutions Inc on Dec 21, 2022.

What is this patent about?

’214 is related to the field of molecular tagging and detection , specifically for identifying and quantifying molecules within individual cells or on particles. The background acknowledges the need for improved methods to analyze gene expression and protein variants at the single-cell level, overcoming limitations of existing techniques that require large sample sizes or lack cell-specific information. The invention aims to address the challenges in multiplexed protein measurement technologies.

The underlying idea behind ’214 is to create a system where each molecule of interest is tagged with a unique identifier that reveals both its identity and its cellular origin. This is achieved by attaching a unique binding agent (UBA) to the target molecule, then linking that UBA to an epitope-specific barcode (ESB) and a cell origination barcode (COB) . The COB is constructed through a split-pool synthesis approach, ensuring each cell receives a unique barcode.

The claims of ’214 focus on a population of beads, where each bead is associated with a nucleic acid tag. This tag includes a cell origination barcode and a degenerate sequence . The key aspect is that different beads have different cell origination barcodes, allowing for the identification of the origin of the molecules attached to the beads. The method claim covers the process of adding these tags to beads using a split-pool barcoding process.

In practice, the invention involves several steps. First, target molecules within cells are bound by UBAs. Next, ESBs are linked to these UBAs. Then, a split-pool synthesis is performed to attach COBs to the ESBs, effectively creating a unique identifier for each target molecule within each cell. Finally, the tagged molecules are detected and quantified, revealing the expression levels of different targets in individual cells.

This approach differs from prior solutions by enabling highly multiplexed analysis at the single-cell level without requiring physical isolation of cells during the tagging process. The use of a split-pool synthesis to generate COBs allows for a large number of unique barcodes to be created, ensuring that each cell is uniquely identified. The addition of a degenerate sequence further enhances the uniqueness and complexity of the tags, improving the accuracy and sensitivity of the analysis.

How does this patent fit in bigger picture?

Technical landscape at the time

In the early 2010s when ’214 was filed, single-cell analysis was an emerging field, at a time when multiplexed protein measurement technologies faced significant challenges due to the complexities inherent in protein samples. Methods for accurate and sensitive detection, identification, and quantification of target molecules in every cell of a complex cell population, while retaining cell-specific information, were still needed. Existing methods often required significant amounts of biological samples or did not provide cell-specific information.

Novelty and Inventive Step

The examiner approved the claims because the prior art before January 31, 2011, did not teach or suggest beads comprising a cell origination barcode and a degenerate sequence (e.g., a random barcode), where each bead has a different cell origination barcode. While some prior art taught beads with barcodes, they failed to teach or suggest that each bead has a different cell origination barcode. Other prior art taught splitting pools of cells containing bead-oligo barcodes but did not teach or suggest that each bead has a different cell origination barcode.

Claims

This patent contains 26 claims, with independent claims 1 and 11. Claim 1 focuses on a population of beads with specific nucleic acid tags, while claim 11 focuses on a method for making barcoded beads. The dependent claims generally elaborate on the composition and characteristics of the beads and nucleic acid tags, as well as specific steps and conditions of the barcoding method.

Key Claim Terms New

Definitions of key terms used in the patent claims.

Term (Source)Support for SpecificationInterpretation
Cell origination barcode
(Claim 1, Claim 11)		“In some embodiments, the invention provides a cell origination barcode (COB). Each COB provides a unique code that can be associated to a specific cell of origin. In some embodiments, upon binding of the COB to a common linker moiety (e.g. common linker oligo) associated with an ESB, the COB code identifies the cells of origin of the target molecule to which the UBA/ESB complex is bound.”A unique identifier associated with a specific cell of origin, used to track the origin of target molecules.
Degenerate sequence
(Claim 1, Claim 11)		“The specification does not provide a specific definition or explanation of 'degenerate sequence'. However, it is understood in the art as a sequence with random or variable nucleotides.”A nucleic acid sequence with random or variable nucleotides, included as part of the nucleic acid tag.
Nucleic acid tag
(Claim 1)		“In some embodiments, the antibodies described herein are attached to a nucleic acid, e.g., linker oligo or a nucleic acid ESB. Methods to attach nucleic acids to antibodies are known in the art. Any suitable method to attach nucleic acids to antibodies is encompassed in the methods of the invention.”A molecule comprising a nucleic acid sequence used to label beads, containing both a cell origination barcode and a degenerate sequence.
Split-pool barcoding process
(Claim 11)		“In some embodiments, during the binding step multiple APSs are added to the tag in an ordered manner during successive rounds of split pool synthesis. In some embodiments, the invention relates to methods for detecting a plurality of properties originating from a plurality of discrete particles, the method comprising: a) providing: i) a population of particles comprising at least a first target molecule; ii) a first unique binding agent (UBA) specific for the first target molecule; iii) a first linkable UBA-dependent epitope specific barcode (ESB); iv) a plurality of round-specific assayable polymer subunit (APS) sets, each set containing a plurality of APSs that are detectably distinct from each other; b) forming at least a first complex comprising said at least first target molecule, said first UBA probe, and said first ESB; c) performing n rounds of split pool synthesis, each round comprising; i) splitting the population of particles into m reaction volumes; ii) contacting one or more reaction volumes with an APS from the APS set specific for the round; iii) pooling two or more reaction volumes; d) detecting a plurality of properties from at least one particle from the population of particles; wherein at least one of the properties relate to a quantity or an identity for a target molecule associated with the particle.”A method of adding nucleic acid tags to beads by dividing the beads into separate pools, barcoding them, and then recombining the pools.

Litigation Cases New

US Latest litigation cases involving this patent.

Case NumberFiling DateTitle
1:25-cv-01287Oct 21, 202510X Genomics, Inc. v. Illumina, Inc.

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US11692214

ROCHE SEQUENCING SOLUTIONS INC
Application Number
US18086383
Filing Date
Dec 21, 2022
Status
Granted
Expiry Date
Jan 31, 2032
External Links
Slate, USPTO, Google Patents