Nucleic Acid Barcoding via Splint-Mediated Ligation
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Solution Overview
Problem
Current methods for processing nucleic acid samples are limited in their ability to efficiently barcode and identify multiple analytes within a biological particle, particularly in a manner that allows for accurate correlation of different nucleic acid molecules.
Innovation Solution
The method involves providing a partition containing a biological particle with specific nucleic acid molecules, including a capture sequence associated with a labelling agent or CRISPR guide RNA, and nucleic acid barcode molecules. Conditions are then provided to extend primer nucleic acid molecules, join them with barcode molecules using a splint sequence, and generate barcoded nucleic acid products.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If multiple nucleic acid molecules are processed separately, then identification accuracy is improved, but processing time and complexity increase
Solution Approach 1:
The patent combines multiple nucleic acid processing operations into a single partitioned reaction environment. Multiple nucleic acid molecules are barcoded and processed simultaneously within the same partition, eliminating the need for separate processing steps while maintaining identification accuracy through the use of unique molecular barcodes for each molecule.
Solution Approach 2:
The patent employs a universal barcoding system that can identify and track multiple different nucleic acid molecules using the same set of reagents and protocols. The barcode molecules serve multiple functions: they identify the original nucleic acid molecule, enable tracking through processing steps, and allow for simultaneous analysis of multiple analytes within a single reaction partition.
2Productivity
If multiple analytes are barcoded simultaneously, then productivity is improved, but barcode correlation accuracy may deteriorate
Solution Approach 1:
The patent segments the barcoding process into distinct molecular components: unique barcodes are attached to specific nucleic acid molecules through controlled ligation reactions. Each barcode molecule is designed with specific sequences that enable selective binding to target nucleic acids, ensuring that even when multiple analytes are processed simultaneously, each barcode remains correctly associated with its target molecule.
Solution Approach 2:
The patent uses splint molecules as intermediaries to facilitate accurate barcode attachment. The splint molecules serve as temporary bridges that hold the barcode and target nucleic acid in proximity during the ligation reaction, ensuring correct pairing. After ligation, the splint is removed, leaving the barcode permanently attached to the correct target molecule without cross-contamination.
3Measurement precision
If partitioned processing is used, then analyte correlation is improved, but device complexity increases
Solution Approach 1:
The patent implements a hierarchical partitioning structure where reactions are organized in nested levels: individual nucleic acid molecules are processed within partitions, which are themselves organized in larger reaction groups. This nested organization allows for systematic tracking and correlation of analytes across multiple processing levels while maintaining a manageable system architecture that can be implemented with standard microfluidic or well-plate technologies.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enables efficient barcoding of multiple nucleic acid molecules within a biological particle, allowing for accurate identification and correlation of different analytes, thereby enhancing the analysis of nucleic acid samples.
Implementation Method 1
extend the primer nucleic acid molecule using the first nucleic acid molecule as a template to generate a first nucleic acid molecule product
Implementation Method 2
join the first nucleic acid molecule product and the first nucleic acid barcode molecule using the splint sequence to generate a first barcoded nucleic acid product
Data Source
AI summary
Provided herein are systems and methods for processing biomolecules (e.g., nucleic acid molecules, proteins) from a sample. A method for processing biomolecules may comprise barcoding the probe-nucleic acid molecule complex or derivatives thereof. Such a method can comprise performing a nucleic acid reaction, e.g., extension, denaturation, and amplification. The methods described herein may be performed within a partition, such as a droplet or well.