In Situ V(D)J Transcript Analysis Using RCA-Enriched cDNA
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Solution Overview
Problem
Current methods struggle to efficiently detect and analyze the spatial distribution of V(D)J transcripts in situ in biological samples, particularly in crosslinked tissues, due to low sensitivity and specificity, and inefficient reverse transcription of V(D)J sequences, leading to loss of sequence information.
Innovation Solution
A method involving reverse transcription of antigen receptor transcripts using primers targeting the constant region, followed by immobilization and circularization of nucleic acid molecules, and subsequent rolling circle amplification (RCA) to enrich and detect V(D)J sequences in situ, using techniques like sequencing by synthesis, ligation, or hybridization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional reverse transcription methods are used to detect V(D)J transcripts, then the detection process is simplified, but sensitivity and specificity are low and sequence information is lost
Solution Approach 1:
The method performs preliminary reverse transcription of V(D)J transcripts into cDNA before detection, using primers that specifically target the constant region. This preliminary action preserves the V(D)J sequence information in the cDNA product, enabling subsequent high-sensitivity and high-specificity detection while avoiding the information loss that occurs with conventional direct detection methods
Solution Approach 2:
The invention introduces cDNA as an intermediary molecule between the V(D)J transcript and the detection process. The cDNA serves as a stable intermediate that preserves sequence information and can be amplified and detected with high sensitivity, resolving the contradiction between detection precision and process complexity
2Loss of information
If V(D)J transcripts are directly detected in situ, then spatial distribution analysis is enabled, but detection sensitivity is insufficient and sequence information is lost
Solution Approach 1:
The method performs preliminary reverse transcription of V(D)J transcripts into cDNA within the tissue section before detection. This preliminary action amplifies the signal while preserving the V(D)J sequence information in the cDNA product, enabling both high sensitivity detection and spatial distribution analysis without information loss
Solution Approach 2:
The invention creates a complementary DNA copy (cDNA) of the V(D)J transcript that preserves the sequence information. This copying process enables subsequent amplification and detection with high sensitivity while maintaining the spatial location information, resolving the contradiction between information preservation and detectable quantity
3Productivity
If conventional detection methods are used, then the process is straightforward, but spatial distribution of V(D)J transcripts cannot be effectively analyzed
Solution Approach 1:
The method performs preliminary reverse transcription and immobilization of cDNA at the original transcript location within the tissue section. This preliminary action creates a stable, amplifiable signal that maintains spatial information, enabling both high-throughput processing and precise spatial distribution analysis of V(D)J diversity
Solution Approach 2:
The cDNA intermediate serves as a bridge that preserves both the sequence information and the spatial location information of the original V(D)J transcript. This intermediary enables subsequent high-throughput amplification and detection while maintaining the precision needed for spatial distribution analysis
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
Enables high-throughput, sensitive, and specific detection of antigen receptor transcripts, preserving sequence information and allowing for spatial analysis of V(D)J diversity in cells and tissues, facilitating improved disease understanding and therapeutic development.
Implementation Method 1
reverse transcribing an antigen receptor transcript at a location in the biological sample
Implementation Method 2
using a polymerase with strand displacement activity
Implementation Method 3
generating a rolling circle amplification (RCA) product of the circularized molecule
Data Source
AI summary
The present disclosure relates in some aspects to methods for analyzing antigen receptor transcripts in a biological sample. In some aspects, nucleic acid molecules are generated from V(D)J transcripts in situ in the biological sample to enrich molecules comprising V(D)J joins. In some aspects, the presence, amount, and/or identity of a plurality of V(D)J transcripts are analyzed in situ. Also provided are oligonucleotides, sets of oligonucleotides, compositions, and kits for use in accordance with the methods.