Covalent cDNA Linking for Immune Repertoire Pairing
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Solution Overview
Problem
Current methods for high-throughput capture of immune repertoire sequences face issues with mispairing and loss of transcripts due to non-covalent attachment of mRNA to capture beads, leading to reduced sensitivity and accuracy in immune cell sequence pairing.
Innovation Solution
The method involves isolating a single cell, lysing it to release mRNA, and then reverse transcribing the mRNA into cDNA that is covalently attached to a solid support within a first container, followed by linking the cDNA molecules in a second container, thereby producing linked nucleic acid molecules that are highly sensitive and reduce transcript loss and mispairing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If mRNA transcripts are non-covalently attached to capture beads, then the capture process is simple and efficient, but transcript loss and mispairing occur due to dehybridization and random binding
Solution Approach 1:
The patent performs reverse transcription to convert mRNA to cDNA before the beads leave the first container. This preliminary conversion ensures that the genetic information is captured in a stable form before any potential transcript loss or mispairing can occur during bead handling and washing steps.
Solution Approach 2:
The patent creates a complementary DNA copy (cDNA) of the mRNA transcript. This copying process preserves the genetic information in a more stable molecule that is less prone to degradation and dehybridization issues, thereby maintaining pairing accuracy through subsequent processing steps.
2Adaptability or versatility
If mRNA transcripts are kept in solution between containers, then the workflow is flexible, but transcripts are vulnerable to degradation by RNases and dehybridization
Solution Approach 1:
The patent creates a complementary DNA copy (cDNA) of the mRNA transcript. This copying process preserves the genetic information in a more stable molecule that is less prone to degradation and dehybridization issues, thereby maintaining pairing accuracy through subsequent processing steps.
Solution Approach 2:
The patent changes the chemical state of the transcript from RNA to DNA through reverse transcription. DNA is inherently more stable than RNA and resistant to RNase degradation, allowing the material to withstand flexible workflow conditions including multiple washing steps and extended processing times without loss of integrity.
3Productivity
If excess transcripts remain free in solution, then capture efficiency does not need to be 100%, but free transcripts can bind randomly to other beads causing mispairing
Solution Approach 1:
The patent performs reverse transcription to convert mRNA to cDNA while the beads are still in the first container, before any washing or re-encapsulation steps. This preliminary action ensures that even if some transcripts are lost or mispaired during handling, the majority that were correctly captured will have their information preserved in cDNA form, maintaining overall pairing fidelity.
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 significantly increases the percentage of correctly linked amplicons from the same cell, improving the fidelity and sensitivity of immune cell receptor sequence pairing, and provides a more robust and flexible workflow.
Implementation Method 1
hybridizing the mRNA molecules to a capture oligonucleotide attached to a solid support, wherein the capture oligonucleotide comprises a sequence complementary to a portion of the mRNA sequence
Implementation Method 2
reverse transcribing the mRNA molecules to produce cDNA molecules in the first container
Implementation Method 3
reverse transcribing the mRNA molecules to produce cDNA molecules in the first container... thereby producing linked nucleic acid molecules that are highly sensitive and reduce transcript loss and mispairing
Data Source
AI summary
Provided are high throughput methods for physically linking cDNA molecules derived from mRNA molecules expressed by the same cell, and libraries of linked cDNA molecules produced by the methods. The methods comprise reverse transcribing mRNA from a single cell in a first container to produce cDNA molecules, and linking the cDNA molecules in a second container. The methods unexpectedly produced libraries of cDNA molecules with an increase in the number of molecules that are correctly linked to other molecules derived from the same cell.