Probe Barcoding for Multiplexed Nucleic Acid Analysis
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Solution Overview
Problem
Current methods for sample processing and analysis, such as PCR and sequencing, face challenges in efficiently processing biological samples, particularly in multiplexed analyses and in minimizing reagent usage.
Innovation Solution
The methods involve hybridizing probes to target nucleic acid molecules, barcoding the probe-molecule complexes, and performing nucleic acid reactions like extension, denaturation, and amplification to generate barcoded nucleic acid molecules, which can be analyzed in partitions like droplets or wells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional PCR and sequencing methods are used for sample processing, then basic nucleic acid analysis can be achieved, but sensitivity and efficiency in multiplexed analyses are insufficient
Solution Approach 1:
The method segments the nucleic acid analysis process into distinct functional modules: probe hybridization to target regions, barcode attachment to probe complexes, and partitioned processing. This segmentation enables simultaneous processing of multiple targets with different probes and barcodes, improving both sensitivity through targeted detection and efficiency through parallel processing in multiplexed analyses
Solution Approach 2:
The patent introduces barcodes as intermediary molecules that bridge the probe-nucleic acid complex and the detection system. These barcodes enable highly sensitive detection by providing amplifiable signals and allow efficient multiplexed analysis by uniquely identifying different probe-target complexes, thereby resolving the contradiction between sensitivity and processing efficiency
2Ease of manufacture
If conventional sequencing methods are used, then nucleic acid sequencing can be performed, but reagent usage is excessive
Solution Approach 1:
The method performs preliminary actions by pre-attaching barcodes to probes before hybridization, and by pre-partitioning samples into discrete reaction volumes. This preliminary preparation reduces reagent consumption during the actual sequencing process by eliminating the need for excessive reagents in subsequent steps, while maintaining ease of manufacture through standardized pre-prepared components
Solution Approach 2:
The patent implements discarding and recovering by using partitioned droplets or wells where unreacted reagents remain confined to their original partitions and can be discarded without affecting other reactions. Simultaneously, the barcoded probe-nucleic acid complexes are recovered and concentrated for sequencing, thereby reducing overall reagent consumption while maintaining ease of operation
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
These methods enable more sensitive and efficient genomic, transcriptomic, or exomic profiling, allow for the detection of variants such as SNPs, and facilitate multiplexed analysis of nucleic acids and proteins while reducing reagent usage.
Implementation Method 1
hybridizing a probe to a molecule of interest (e.g., target protein, target nucleic acid molecule)
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 hybridizing a probe molecule to a target region of a nucleic acid molecule (e.g., a ribonucleic acid (RNA) molecule) and 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. A method for processing a sample may comprise hybridizing probes to (i) target regions of a nucleic acid molecule (e.g., RNA molecule) and (ii) a reporter oligonucleotide of a feature binding group, and barcoding the probe-associated molecules. One or more processes of the methods described herein may be performed within a partition, such as a droplet or well.