Nested PCR Cloning of Single-Cell TCR Repertoires
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Solution Overview
Problem
Current methods for cloning functional T cell receptors (TCRs) from single T cells are inefficient and miss a significant percentage of possible α/β or γ/δ variable chain combinations, making it difficult to obtain a comprehensive repertoire of TCRs for therapeutic applications such as cancer therapies.
Innovation Solution
A method involving nested amplification procedures using specific primer collections to amplify and clone nucleic acid encoding TCRs from single T cells, allowing for the generation of nucleic acid vectors that express functional TCRs with high accuracy and efficiency, including the VJ and VDJ segments, enabling the cloning of hundreds to thousands of TCRs simultaneously with minimal missing rates.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional cloning methods are used, then the process is simpler, but the coverage of TCR repertoire is incomplete (significant percentage missed)
Solution Approach 1:
The patent segments the TCR cloning process into distinct phases: (1) single T cell isolation and lysis, (2) cDNA synthesis from single cell RNA, (3) nested PCR amplification with specific primer collections for different TCR chains, (4) cloning into expression vectors, and (5) transformation into E. coli. This segmentation allows systematic optimization of each step to maximize repertoire coverage while managing complexity.
Solution Approach 2:
The patent employs nested PCR amplification where outer primers first amplify larger TCR gene segments, followed by inner primers that amplify specific regions of interest. This nested approach enables comprehensive coverage of diverse TCR variable regions while maintaining amplification efficiency and reducing false negatives.
2Reliability
If comprehensive primer collections are used to cover all TCR variable regions, then the TCR repertoire coverage improves, but the cost and time required increases
Solution Approach 1:
The patent performs preliminary actions by designing and preparing comprehensive primer collections in advance that target all known TCR variable regions. These primers are pre-synthesized and ready for use, eliminating the need for time-consuming primer design and optimization during the actual cloning process. The primers are also pre-tested for specificity and efficiency.
Solution Approach 2:
The patent implements continuous amplification through nested PCR where the first round amplifies TCR chains and the second round immediately amplifies specific regions for cloning. This continuous process minimizes idle time between steps and maintains reaction efficiency throughout the procedure.
3Productivity
If nested amplification procedures with specific primer collections are used, then the cloning efficiency and accuracy improve, but the procedural complexity increases
Solution Approach 1:
The patent develops universal primer collections that can amplify all human TCR variable regions (TRAV1-45, TRBV1-50, TRGV1-10, TRDV1-10) using the same protocol. These universal primers contain conserved regions that bind to diverse TCR sequences, allowing a single set of primers to function across the entire TCR repertoire without requiring separate amplification protocols for different TCR types.
Solution Approach 2:
The patent optimizes PCR parameters including annealing temperatures, extension times, and cycle numbers to maximize amplification efficiency across all TCR variable regions. By carefully adjusting these parameters, the nested amplification procedure achieves high productivity while managing procedural complexity through standardized conditions.
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 the rapid and effective cloning of functional TCRs from single T cells, enabling the generation of expression vectors that accurately represent the TCR repertoire, facilitating the identification of rare TCRs and their use in therapeutic applications like cancer therapies with high effectiveness and efficiency.
Implementation Method 1
performing a nested polymerase chain reaction (PCR) amplification procedure using the cDNA of each of the plurality of separate locations as template to obtain a first amplification product and a second amplification product
Implementation Method 2
performing a reverse transcription reaction to obtain a cDNA from the RNA
Data Source
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AI summary
This document provides methods and materials involved in cloning functional TCRs from single T cells. For example, methods and materials for obtaining nucleic acid encoding a TCR from a single T cell and arranging that nucleic acid to form nucleic acid vectors successfully designed to express a TCR, kits for obtaining nucleic acid encoding a TCR from a single T cell and arranging that nucleic acid to form nucleic acid vectors successfully designed to express a TCR, methods for making such kits, collections of nucleic acid primers designed to amplify the entire coding sequence of both variable regions for each expressed V segment for functional αβ or γδ TCRs of a particular mammalian species, methods for using such collections of nucleic acid primers to clone functional TCRs from single T cells, and kits containing such collections of nucleic acid primers to clone functional TCRs from single T cells are provided.