Nucleic Acid Capture With Simultaneous RT–T4 Ligation
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Solution Overview
Problem
Current spatial transcriptomic methods fail to capture a sufficient number of genes, leading to inaccuracies in gene expression analysis due to cDNA loss, which hinders the reflection of true gene expression in tissues.
Innovation Solution
A method combining reverse transcription and T4 ligation into a single step, using splint hybridization to ligate cDNA to an oligonucleotide strand on a capture chip, optimizing reaction conditions to enhance gene capture efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If reverse transcription and T4 ligation are performed as separate sequential steps, then each step can be optimized independently, but the total process duration is extended and cDNA loss increases
Solution Approach 1:
The patent combines reverse transcription and T4 ligation into a single simultaneous reaction step. The reaction system includes both reverse transcriptase and T4 ligase working together in the same buffer at 37°C for 3-5 hours, allowing cDNA synthesis and ligation to the capture chip to occur concurrently, thereby reducing total process time and minimizing cDNA loss.
Solution Approach 2:
The patent performs preliminary hybridization of the random probe to the RNA target and preparation of the reaction mixture before initiating the simultaneous RT-ligation reaction. The splint oligonucleotide is pre-hybridized to the capture chip, and all reaction components are prepared in advance to ensure immediate onset of the combined reaction, maximizing efficiency.
2Reliability
If reverse transcription and T4 ligation are performed as separate sequential steps, then reaction conditions can be independently optimized, but cDNA loss occurs during the transition between steps
Solution Approach 1:
The patent combines reverse transcription and T4 ligation into a single simultaneous reaction step. The reaction system includes both reverse transcriptase and T4 ligase working together in the same buffer at 37°C for 3-5 hours, allowing cDNA synthesis and ligation to the capture chip to occur concurrently, thereby reducing total process time and minimizing cDNA loss.
Solution Approach 2:
The patent ensures continuous action by maintaining the cDNA in the reaction mixture throughout the process. As cDNA is synthesized by reverse transcriptase, it is immediately available for ligation by T4 ligase to the capture chip, eliminating any intermediate steps where cDNA could be lost or degraded. The reaction proceeds continuously without interruption or transfer.
3Reliability
If multiple separate steps are used for RNA capture, then each step can be controlled independently, but the overall process complexity increases
Solution Approach 1:
The patent combines reverse transcription and T4 ligation into a single simultaneous reaction step. The reaction system includes both reverse transcriptase and T4 ligase working together in the same buffer at 37°C for 3-5 hours, allowing cDNA synthesis and ligation to the capture chip to occur concurrently, thereby reducing total process time and minimizing cDNA loss.
Solution Approach 2:
The patent employs a universal reaction buffer that supports both reverse transcription and T4 ligation activities. The buffer composition is optimized to accommodate both enzymes simultaneously, creating a multi-functional reaction system that performs both cDNA synthesis and ligation in one step, reducing the number of separate reagent systems needed.
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
Significantly increases the number of captured genes and reduces cDNA loss, while significantly shortening the process duration, improving gene capture efficiency by up to 6.5-fold compared to existing methods.
Implementation Method 1
a random probe such as 6N, and a splint are hybridized in 5× SSC buffer at 55°C for 10 minutes
Implementation Method 2
reverse transcribing an RNA captured in tissues into a stable cDNA
Implementation Method 3
the cDNA is ligated, by means of splint hybridization of fixed oligonucleotide sequences at both ends, to an oligonucleotide strand fixed on the chip
Implementation Method 4
the cDNA is ligated, by means of splint hybridization of fixed oligonucleotide sequences at both ends
Data Source
Figure 1(1)~2(3)
Figure 3
Figure 4(1)~5(2)
AI summary
An improved nucleic acid capture method, comprising the following steps: by means of a splint oligonucleotide hybridization random probe and an oligonucleotide strand fixed on a capture chip, an RNA in a sample captured by the random probe undergoing reverse transcription into a cDNA, and linking the cDNA to the oligonucleotide strand, the reverse transcription and the linking being carried out in the same reaction system. During the process, while the RNA undergoes the reverse transcription into the cDNA, the cDNA is linked, by means of splint hybridization of fixed oligonucleotide sequences at two ends, to the oligonucleotide strand fixed on the chip. The method can increase the number of captured genes, and reduce the loss of cDNA; in addition, the method combines what was originally three steps into one step, thus further shortening the duration of the process.