Analysis Of Nucleic Acid Sequences
Patent No. US12275993 (titled "Analysis Of Nucleic Acid Sequences") was filed by 10X Genomics Inc on Oct 28, 2024.
What is this patent about?
’993 is related to the field of single-cell analysis and, more specifically, to methods for processing droplets containing cells or nuclei for downstream analysis, such as sequencing. The background involves partitioning biological samples into droplets for separate processing, which allows for accurate control of the reaction environment. A challenge exists in changing the amount of reagent in a droplet to affect the rate or progress of a reaction within the droplet.
The underlying idea behind ’993 is to use micelles as carriers to transfer a lysis reagent from a first population of droplets to a second population. The first droplet population contains the lysis reagent, while the second contains cells or nuclei and nucleic acid barcode molecules. This transfer enables controlled lysis of the cells or nuclei within the second droplet population, facilitating downstream analysis.
The claims of ’993 focus on a method that involves providing a first emulsion containing droplets with a lysis reagent and a second emulsion containing droplets with cells or nuclei and nucleic acid barcode molecules. The method then subjects the two emulsions to conditions that allow the lysis reagent to be transferred to the second droplet population via micelles. Finally, the method lyses the cells or nuclei within the droplets of the second population using the transferred lysis reagent.
In practice, the method involves creating two separate emulsions. The first emulsion contains droplets with a high concentration of a lysis reagent, such as n-dodecyl-beta-D-maltoside (DBDM). The second emulsion contains cells or nuclei, along with beads that are coupled to nucleic acid barcode molecules. When these two emulsions are mixed, the lysis reagent is transferred from the first droplet population to the second via micelles. This transfer process is driven by the concentration gradient and the ability of micelles to solubilize and transport the lysis reagent.
This approach differs from prior methods by providing a mechanism to control the concentration of lysis reagent in the droplets containing cells or nuclei. Instead of directly introducing the lysis reagent into the cell-containing droplets, it is transferred via micelles from a separate droplet population. This allows for a more controlled and efficient lysis process, which is crucial for preserving the integrity of the nucleic acids and ensuring accurate downstream analysis, such as barcoding and sequencing.
How does this patent fit in bigger picture?
Technical landscape at the time
In the late 2010s, at a time when biological samples were commonly processed using PCR and sequencing techniques, partitioning reagents in droplets was a known method for analyzing samples. However, controlling the amount of reagent within a droplet to influence reaction rates was a non-trivial challenge, often requiring precise microfluidic systems or complex chemical manipulations.
Novelty and Inventive Step
The examiner approved the application because the prior art did not teach or suggest a method of transferring a lysis reagent between a first emulsion comprising a first droplet population and a second emulsion comprising a second droplet population via micelles. Specifically, the prior art failed to disclose subjecting the first and second emulsions to conditions sufficient to transfer the lysis reagent via micelles to lyse cells or nuclei within the second droplet population, where the second droplet population also contains nucleic acid barcode molecules. The examiner also stated that the prior art did not teach or suggest generating a first emulsion with droplets and micelles containing a chemical lysis agent, then contacting this emulsion with a second emulsion to transfer the lysis agent via micelles to the second droplet population to lyse cells or nuclei containing target nucleic acids and particles coupled to nucleic acid barcode molecules.
Claims
This patent contains 30 claims, with claims 1 and 25 being independent. The independent claims are directed to methods involving the lysis of cells or nuclei within droplets of an emulsion using a lysis reagent transferred via micelles. The dependent claims generally elaborate on and refine the elements and steps of the independent claims, providing more specific details regarding the composition of the emulsions, the lysis reagents, and the process of generating barcoded nucleic acid molecules.
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Date
Description
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US12275993
- Application Number
- US18929514
- Filing Date
- Oct 28, 2024
- Status
- Granted
- Expiry Date
- Feb 11, 2040
- External Links
- Slate, USPTO, Google Patents