Stochastic Barcode TCR Labeling for Cost-Effective Immunological Analysis
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Solution Overview
Problem
Existing methods for stochastic labeling of nucleic acids are costly and inefficient, particularly for specific targets like the T cell receptor (TCR) alpha and beta chains, as they require expensive reagents and complex designs.
Innovation Solution
The method involves hybridizing target nucleic acids with oligonucleotides containing universal and target-specific sequences, extending these sequences, and incorporating stochastic barcodes for amplification, allowing for efficient labeling and quantification of TCR alpha and beta chains using a combination of amplification primers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If traditional stochastic labeling methods are used for TCR alpha and beta chains, then labeling can be achieved, but the cost is high and the process is inefficient due to expensive reagents and complex designs
Solution Approach 1:
The patent employs universal adaptor sequences that can bind to multiple different TCR chains (both alpha and beta chains) through a common constant region. This universal adaptor allows a single stochastic barcode system to label different TCR types, eliminating the need for separate expensive reagents for each TCR chain and achieving cost-effective multi-target labeling while maintaining reliability through consistent universal binding
2Measurement precision
If target-specific reagents are designed for each TCR chain, then specific labeling is achieved, but the design complexity and cost increase significantly
Solution Approach 1:
The TCR labeling system is segmented into two functional parts: a universal adaptor sequence that binds to the constant region (common to all TCRs) and a variable region that provides target specificity. This segmentation allows the complex specific binding function to be separated from the universal labeling function, reducing overall system complexity while maintaining both specificity and universality
Solution Approach 2:
The universal adaptor sequence acts as an intermediary between the stochastic barcode system and the diverse TCR targets. Instead of designing specific reagents for each TCR chain, the adaptor serves as a universal mediator that bridges the barcode system to all TCR types through their common constant region, dramatically simplifying the reagent design while preserving target specificity through the adaptor's selective binding
3Productivity
If multiple amplification primers are used for TCR characterization, then comprehensive analysis is achieved, but the amplification process becomes more complex
Solution Approach 1:
The patent merges the universal adaptor sequence into the stochastic barcode construct, creating a unified amplifiable unit that contains both the barcode and the universal binding site. This merging allows a single primer set to amplify all barcoded TCR products simultaneously, combining multiple functions into one streamlined amplification process that maintains comprehensive analysis capability while reducing procedural complexity
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 cost-effective and efficient labeling and quantification of TCR alpha and beta chains, facilitating the characterization of T cells by using stochastic barcodes to distinguish and count individual targets within a sample.
Implementation Method 1
hybridizing a target with a first oligonucleotide comprising a universal adaptor sequence and a target-specific sequence
Implementation Method 2
extending said first oligonucleotide to generate a first nucleotide sequence
Implementation Method 3
hybridizing said first nucleotide sequence with a second oligonucleotide
Implementation Method 4
extending said second oligonucleotide to generate a second nucleotide sequence
Implementation Method 5
hybridizing said second nucleotide sequence with a third oligonucleotide comprising a binding site for a first amplification primer and the universal adaptor sequence
Implementation Method 6
extending said third oligonucleotide to generate a third nucleotide sequence
Implementation Method 7
amplifying said third nucleotide sequence using a first amplification primer to generate a plurality of a fourth nucleotide sequences
Data Source
AI summary
Embodiments provided herein relate to methods and compositions for immunological analysis. Some embodiments relate to methods and compositions for characterizing a sample by identifying the TCR alpha chain or beta chain of a T cell.