Analysis Of Nucleic Acid Sequences
Patent No. US12305239 (titled "Analysis Of Nucleic Acid Sequences") was filed by 10X Genomics Inc on Nov 18, 2024.
What is this patent about?
’239 is related to the field of droplet processing, specifically methods for manipulating the concentration of reagents within droplets used for biological assays. The background involves challenges in changing reagent amounts within droplets to control reaction rates, particularly in applications like single-cell analysis where precise control over lysis and barcoding is crucial. Existing methods lack efficient ways to adjust reagent concentrations post-droplet formation.
The underlying idea behind ’239 is to leverage mass transport phenomena to alter reagent concentrations in droplets after they have been formed. This is achieved by creating two droplet populations: one with a high concentration of a reagent (e.g., a lysis agent) and another containing cells and barcoding beads. By facilitating the transfer of the reagent between these populations, the cells can be lysed and their contents barcoded in a controlled manner.
The claims of ’239 focus on a method involving: generating a first set of droplets containing a lysis agent; partitioning cells and barcoding molecules into a second set of droplets; forming a micellized lysis agent from the first droplet type; delivering the lysis agent to the second droplet type using the micellized form; lysing the cells; hybridizing mRNA to the barcoding molecules; and generating barcoded nucleic acids. A key aspect is the use of micelles to transport the lysis agent.
In practice, the invention involves creating a water-in-oil emulsion where some droplets contain a high concentration of a lysis agent, such as n-dodecyl-beta-D-maltoside (DBDM), while other droplets contain cells and barcoded beads. The lysis agent is then transferred from the first droplet population to the second via micelle-mediated diffusion . This allows for controlled cell lysis within the second droplet population, releasing mRNA for subsequent barcoding and sequencing.
This approach differs from prior methods by enabling post-droplet formation control of reagent concentration. Instead of relying on precise initial reagent loading, ’239 uses mass transport to adjust the chemical environment within the droplets. This is particularly useful for applications where a high initial reagent concentration might be detrimental, or where precise control over the timing and extent of lysis is required for optimal downstream analysis, such as in single-cell RNA sequencing.
How does this patent fit in bigger picture?
Technical landscape at the time
In the late 2010s when ’239 was filed, single-cell analysis at a time when single-cell analysis was typically implemented using microfluidic devices to partition cells into droplets. At this time, barcoding nucleic acids within droplets was a common approach for identifying the origin of genetic material from individual cells. Systems commonly relied on precise control of reagent concentrations within these droplets to ensure efficient cell lysis and subsequent reactions. Hardware or software constraints made precise control of reagent concentrations within droplets non-trivial.
Novelty and Inventive Step
The examiner allowed the claims because the prior art failed to teach or suggest a method that combines generating a first set of droplets containing a lysis agent, partitioning cells into a second set of droplets containing cells and barcoded molecules, and generating a micellized lysis agent from the first droplet to deliver the lysis agent to the second droplet. The examiner also stated that the prior art does not disclose or suggest generating a micellized lysis agent from the first droplet, where the micellized lysis agent comprises a micelle comprising the lysis agent and delivering the lysis agent to the second droplet, as well as lysing the cell within the second droplet with the lysis agent, thereby releasing a messenger RNA (mRNA) molecule from the cell into the second droplet, as disclosed and claimed by the instant application. The examiner also stated that the prior art does not disclose or suggest generating a micellized lysis agent from the first droplet, wherein the micellized lysis agent comprises a micelle comprising the lysis agent and delivering the lysis agent to the second droplet using the micellized lysis agent as well as lysing the cell within the second droplet with the lysis agent, thereby releasing a messenger RNA (mRNA) molecule from the cell into the second droplet, as disclosed and claimed by the instant application.
Claims
This patent contains 30 claims, with independent claims 1 and 29. The independent claims are directed to methods of generating barcoded nucleic acid molecules from cells using droplet microfluidics and a micellized lysis agent. The dependent claims generally elaborate on specific aspects, conditions, or components of the methods described in the independent claims.
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Date
Description
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US12305239
- Application Number
- US18951533
- Filing Date
- Nov 18, 2024
- Status
- Granted
- Expiry Date
- Feb 11, 2040
- External Links
- Slate, USPTO, Google Patents