Single-Cell Nucleic Acid Detection with Unique Molecular Barcodes
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Solution Overview
Problem
Existing methods struggle to provide genome-wide, digital quantification of nucleic acid molecules with high dynamic range and single molecule sensitivity, particularly for low copy number RNA and DNA detection in single cells, due to amplification bias and low sensitivity.
Innovation Solution
A method involving unique barcode sequences is used to tag and label nucleic acid molecules, followed by amplification and sequencing, ensuring each molecule has a distinct barcode, allowing for accurate digital counting and reducing amplification bias.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional nucleic acid detection methods are used, then the detection process is simple, but the sensitivity is insufficient for low copy number molecules and amplification bias occurs
Solution Approach 1:
The invention segments the nucleic acid detection process into distinct stages: barcoding individual molecules, amplification, and sequencing. By assigning unique barcodes to individual molecules before amplification, the method enables precise counting of low copy number molecules while maintaining simplicity through modular process design
Solution Approach 2:
The invention applies preliminary barcoding action to individual nucleic acid molecules before amplification. This preliminary tagging with unique barcodes allows subsequent digital counting to accurately quantify low copy number molecules, resolving the sensitivity issue without requiring complex detection mechanisms
2Measurement precision
If amplification is performed to detect low copy number molecules, then detection sensitivity improves, but amplification bias is introduced
Solution Approach 1:
The invention uses digital copying through barcode sequencing to count individual molecules. Instead of relying on analog signal amplification that introduces bias, the method creates digital copies of molecule identities through unique barcodes, enabling accurate quantification without amplification bias
Solution Approach 2:
The invention introduces unique barcodes as intermediary elements between the original nucleic acid molecules and the detection system. These barcodes serve as mediators that preserve molecular identity through amplification, allowing accurate counting while eliminating direct amplification bias on the target molecules
3Measurement precision
If unique barcode sequencing is used for digital counting, then single molecule sensitivity is achieved, but the process becomes more complex
Solution Approach 1:
The invention segments the complex digital counting process into three simple sequential steps: barcoding individual molecules, amplifying the barcoded molecules, and sequencing to read barcodes. This segmentation achieves single molecule sensitivity while keeping each individual step simple and manageable
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 precise quantification of nucleic acid molecules, including low copy numbers, with high sensitivity and reduced bias, facilitating comprehensive expression profiling of single cells.
Implementation Method 1
A method of identifying target molecules in a sample, such as a plurality of target molecules from a single cell, using unique barcode sequences
Implementation Method 2
The tagged nucleic acid molecules with their own unique barcode sequences are then amplified in the case of DNA, such as cDNA
Implementation Method 3
The amplicons are then sequenced whether produced from DNA or RNA and the barcodes are identified
Data Source
AI summary
Methods and compositions for digital profiling of nucleic acid sequences present in a sample are provided.