Single-Cell Nucleic Acid Profiling With Barcode Counting
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods struggle to provide a technique for genome-wide, digital quantification of nucleic acid molecules with high dynamic range and single molecule sensitivity, especially for low copy number RNA and DNA detection in single cells.
Innovation Solution
A method involving unique barcode sequences is used to tag and label nucleic acid molecules, followed by amplification and sequencing to count the molecules accurately, reducing amplification bias and enabling high sensitivity counting.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional nucleic acid detection methods are used, then detection can be performed, but sensitivity is insufficient for low copy number molecules and dynamic range is limited
Solution Approach 1:
The patent segments the detection process into distinct stages: unique barcode tagging of individual molecules, selective amplification of barcoded molecules, and sequencing. This segmentation allows each stage to be optimized independently, with the barcode providing a handle for tracking individual molecule origins through the workflow, thereby achieving single-molecule sensitivity across a wide dynamic range.
Solution Approach 2:
The unique barcode sequence acts as an intermediary element between the target nucleic acid molecules and the detection system. The barcode is attached to each molecule, allowing indirect detection through amplification and sequencing of the barcode rather than direct detection of the target, which enables sensitive detection of low copy number molecules.
2Measurement precision
If amplification is performed to increase detection sensitivity, then sensitivity improves, but amplification bias is introduced
Solution Approach 1:
The patent performs preliminary action by attaching unique barcodes to individual nucleic acid molecules before amplification. This preliminary tagging ensures that each molecule is individually identified and tracked, allowing subsequent amplification to proceed without introducing bias in the quantification, since the barcode count directly reflects the original molecule number.
Solution Approach 2:
The patent uses copying by creating multiple copies of the barcode sequence through amplification, while the barcode itself serves as a unique identifier. This allows the target molecules to be amplified for detection while the barcode information is preserved and counted, enabling sensitive detection without amplification bias in the quantification.
3Adaptability or versatility
If genome-wide expression profiling is performed, then comprehensive data is obtained, but complexity and difficulty of detection increase
Solution Approach 1:
The patent applies universality by using a universal barcode tagging approach that can be applied to all nucleic acid molecules in the sample simultaneously. The same barcode attachment and amplification protocol works for all genes and molecules, enabling genome-wide profiling without increasing procedural complexity, as the unique barcode system scales uniformly across the entire transcriptome.
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
The method allows for accurate digital counting of nucleic acid molecules with high sensitivity and reduced bias, revealing low copy number RNA and DNA in single cells.
Implementation Method 1
The amplicons are then sequenced whether produced from DNA or RNA and the barcodes are identified
Implementation Method 2
Each cDNA is then amplified to produce amplicons of the cDNA
Data Source
AI summary
Methods and compositions for digital profiling of nucleic acid sequences present in a sample are provided.