Probe-Based RNA Barcoding Without Reverse Transcription
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Solution Overview
Problem
Current methods for processing and analyzing biological samples, such as PCR and sequencing, often require reverse transcription and are inefficient in controlling reaction environments within partitions like droplets or wells.
Innovation Solution
The method involves hybridizing probes to target regions of nucleic acid molecules, linking the probes, and barcoding the resultant complex within a partition, thereby obviating the need for reverse transcription and enabling controlled analysis and processing of analytes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If reverse transcription is used in current PCR and sequencing methods, then RNA analysis is enabled, but the process complexity and time consumption increase
Solution Approach 1:
The patent extracts and eliminates the reverse transcription step from the traditional PCR/sequencing workflow. By using probe-based hybridization methods that directly target RNA molecules, the invention removes the need for reverse transcription while maintaining RNA analysis capability, thereby simplifying the process and improving efficiency
Solution Approach 2:
Instead of converting RNA to cDNA through reverse transcription and then amplifying, the invention inverts the approach by using probes that directly hybridize to RNA targets and enable amplification without the intermediate reverse transcription step, fundamentally changing the workflow sequence
2Reliability
If biological samples are processed in partitions like droplets or wells, then controlled reaction environments are achieved, but the ability to accurately control and monitor reaction conditions within each partition is reduced
Solution Approach 1:
The patent introduces barcodes as intermediary elements that are attached to nucleic acid molecules within partitions. These barcodes serve as mediators that carry information about the partition identity and reaction conditions, enabling accurate tracking and control of reactions within each partition without requiring complex monitoring systems
Solution Approach 2:
The invention uses barcode sequences with varying parameters (different nucleotide compositions, lengths, or patterns) to encode information about reaction conditions and partition identities. By changing barcode parameters rather than physical partition characteristics, the system achieves precise control and monitoring capability
3Measurement precision
If probes are hybridized to target regions and linked to form probe-linked nucleic acid molecules, then specific nucleic acid sequences can be identified, but the process requires additional steps and time
Solution Approach 1:
The patent merges multiple functions into the probe structure itself. The probes are designed to simultaneously perform hybridization to target sequences, serve as primers for amplification, and carry barcode sequences for identification. This consolidation eliminates separate steps for each function, reducing overall processing time while maintaining precision
Solution Approach 2:
The invention performs preliminary barcoding of probes before hybridization. By pre-attaching barcode sequences to probes, the system eliminates the need for post-hybridization barcode attachment steps, thereby reducing processing time while ensuring accurate sequence identification through the pre-integrated barcodes
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 allows for efficient analysis and processing of nucleic acid molecules without reverse transcription, facilitating accurate control of reaction environments within partitions and generating amplified products for further analysis.
Implementation Method 1
hybridize the first probe sequence of the first probe to the first target region of the nucleic acid molecule
Implementation Method 2
subjecting the first reactive moiety and the second reactive moiety to conditions sufficient to yield a probe-linked nucleic acid molecule comprising the first probe linked to the second probe
Data Source
AI summary
The present disclosure provides methods of processing or analyzing a sample. A method for processing a sample may comprise hybridizing a probe molecule to a target region of a nucleic acid molecule (e.g., a ribonucleic acid (RNA) molecule), barcoding the probe-nucleic acid molecule complex, and performing extension, denaturation, and amplification processes. A method for processing a sample may comprise hybridizing first and second probes to adjacent or non-adjacent target regions of a nucleic acid molecule (e.g., an RNA molecule), linking the first and second probes to provide a probe-linked nucleic acid molecule, and barcoding the probe-linked nucleic acid molecule. One or more processes of the methods described herein may be performed within a partition, such as a droplet or well. One or more processes of the methods described herein may be performed on a cell, such as a permeabilized cell.