TCR cDNA Amplification Using Segmented Primer Strategy
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Solution Overview
Problem
Current methods for amplifying T cell receptor (TCR) genes in TCR gene therapy are inefficient, requiring multiple steps and significant time, especially when trying to isolate cancer-specific TCRs from peripheral blood lymphocytes, as they often necessitate the establishment of T cell clones and involve complex PCR processes that complicate analysis and increase costs.
Innovation Solution
A method using 80 kinds of primers corresponding to leader sequences with a translation initiation codon for RT-PCR, allowing for efficient amplification of TCR cDNA from a single T cell, including a leader sequence, which simplifies the process by reducing the need for additional steps and minimizing material costs, enabling rapid isolation of cancer-specific TCRs without the need for T cell cloning.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If the 5'-RACE method is used to amplify TCR cDNA, then the leader sequence is included in the amplification product, but cDNAs of various lengths are amplified making subsequent analysis complicated and additional time and material cost are required
Solution Approach 1:
The patent segments the amplification process into two distinct PCR steps: first amplifying TCR cDNA with leader sequence using 5'-RACE method, then performing a second PCR to selectively amplify only the desired length products. This segmentation allows the complex task of obtaining precise amplification products to be broken down into manageable steps, resolving the contradiction between including leader sequence and maintaining analysis simplicity.
Solution Approach 2:
The patent introduces an intermediary step (second PCR) that acts as a filter between the initial amplification and final analysis. This intermediary PCR step with specific primers selectively amplifies only the cDNA products of the desired length range, eliminating the complexity of analyzing various length products while preserving the leader sequence inclusion benefit.
2Manufacturing precision
If the 5'-RACE method is used to amplify TCR cDNA, then the leader sequence is included, but additional steps and material costs are required compared to common RT-PCR
Solution Approach 1:
The patent performs preliminary action by designing and preparing specific primers in advance that can directly amplify TCR cDNA with leader sequence in a single RT-PCR step. This preliminary preparation of optimized primers eliminates the need for the time-consuming 5'-RACE procedure while ensuring the leader sequence is included in the amplification product.
3Productivity
If common RT-PCR is used to amplify TCR cDNA, then the procedure is simpler and faster, but cDNA containing neither translation initiation codon nor leader sequence is amplified requiring additional steps for gene expression
Solution Approach 1:
The patent makes the amplification procedure multi-functional by designing primers that simultaneously achieve multiple objectives: amplifying TCR cDNA efficiently, including the leader sequence, and incorporating the translation initiation codon. This universal primer design eliminates the need for separate steps to add these essential elements, resolving the contradiction between amplification efficiency and expression preparation ease.
4Reliability
If T cell cloning is performed to obtain cancer-specific TCR, then the specificity can be verified, but the procedure requires several months
Solution Approach 1:
The patent extracts the essential function of T cell cloning (obtaining cancer-specific TCR) and performs it directly at the molecular level by amplifying TCR cDNA from single T cells using specific primers. This extraction eliminates the time-consuming cellular cloning and proliferation steps while maintaining the ability to verify cancer-specificity through direct sequencing of the amplified TCR genes.
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 method allows for the efficient amplification of TCR cDNA with a complete leader sequence from a single T cell, significantly reducing the time required for TCR gene isolation to just a few days and eliminating the need for T cell cloning, thereby streamlining the TCR gene therapy process.
Implementation Method 1
cDNAs produced from mRNAs by using a reverse transcriptase
Implementation Method 2
If PCR is performed with such cDNAs, cDNAs of various lengths are amplified
Data Source
AI summary
As a method for highly efficiently amplifying a TCR cDNA in a short period of time, there is provided a method for amplifying a T cell receptor (TCR) cDNA, which comprises the following step (1) and step (2):(1) the step of performing PCR by using at least one kind of the L primer mentioned below, the C primer 1 or UTR primer 1 mentioned below, and cDNA obtained from a single cell as the template to obtain an amplification product 1;an L primer of 30- to 60-nucleotide length comprising an adapter part of 15- to 25-nucleotide length, and a leader region-annealing part of 15- to 25-nucleotide length, which is ligated downstream from the adapter part, and can anneal to a part of a leader region containing a translation initiation codon, or an upstream part thereof,a C primer 1 of 15- to 25-nucleotide length, which can anneal to a part of a constant region, or a UTR primer 1 of 15- to 25-nucleotide length, which can anneal to a part of a 3′ untranslated region;(2) the step of performing PCR by using the adaptor primer mentioned below, the C primer 2 or UTR primer 2 mentioned below, and the amplification product 1 as the template to obtain an amplification product 2;an adapter primer of 15- to 25-nucleotide length, which can anneal to the adapter part of the amplification product 1,a C primer 2 of 15- to 25-nucleotide length, which can anneal to a part of the constant region existing upstream from the region to which the C primer 1 anneals, or a UTR primer 2 of 15- to 25-nucleotide length, which can anneal to a part of the 3′ untranslated region existing upstream from the region to which the UTR primer 1 anneals.