5′-Uracil Splint Sequence for One-Pot Nucleic Acid Capture
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Solution Overview
Problem
Existing nucleic acid capture methods using random probes result in the loss of captured cDNA information due to the separation of reverse transcription (RT) and T4 ligation into two steps, requiring different temperature equipment and being time-consuming, leading to reduced sequencing data mapping rates and increased costs.
Innovation Solution
A splint sequence with a 5' end uracil (U) is used to prevent hybridization with sequencing adapters, allowing simultaneous reverse transcription and ligation in a single reaction system, reducing the generation of splint-induced adapters.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If RT and T4 ligation are separated into two steps, then the ligation reaction can be performed, but the process becomes time-consuming and requires two types of constant temperature equipment
Solution Approach 1:
The patent combines RT and T4 ligation into a single reaction step by using a splint sequence that allows both reactions to occur simultaneously in one pot at 42°C, eliminating the need for separate equipment and steps
2Reliability
If RT and T4 ligation are separated into two steps, then each reaction can be optimized, but the total reaction time increases
Solution Approach 1:
The patent merges RT and T4 ligation into a single simultaneous reaction process occurring at 42°C, reducing total reaction time from overnight plus 3 hours to just 3 hours while maintaining reaction efficiency through the splint sequence design
3Reliability
If the fixed oligonucleotide sequence on the random probe hybridizes with the splint sequence, then ligation can occur, but splint-induced adapters are generated reducing sequencing data mapping rates
Solution Approach 1:
The patent extracts the problematic 5' end portion of the splint sequence that causes unwanted hybridization, replacing it with a modified sequence that eliminates adapter generation while preserving the necessary ligation function
Solution Approach 2:
The patent modifies the splint sequence parameters by changing the 5' end nucleotide composition to prevent hybridization with the fixed oligonucleotide sequence, thereby stopping the formation of splint-induced adapters and improving sequencing data mapping rates
4Ease of manufacture
If a known fixed oligonucleotide sequence is added to the 3' end of cDNA, then sequencing adapters are formed, but the oligonucleotide strand on the capture chip becomes useless and cDNA is lost
Solution Approach 1:
The patent removes the harmful hybridization interaction between the fixed oligonucleotide sequence and splint by modifying the splint's 5' end, extracting the problematic binding capability while retaining the ligation function, thus preventing cDNA loss
Solution Approach 2:
The patent converts the potential harm of splint hybridization into a benefit by designing a modified splint sequence that specifically avoids hybridizing with the fixed oligonucleotide, thereby preventing adapter generation and cDNA loss while maintaining ligation efficiency
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
Improves sequencing data mapping rates and reduces sequencing costs by minimizing the number of adapters generated, enhancing the efficiency of nucleic acid capture.
Implementation Method 1
the oligonucleotide sequence fixed on the chip, after extension using the splint sequence as a template, fails to hybridize with the TSO at the 5' end of the splint sequence
Implementation Method 2
random probes (e.g., 6N) are generally used in the prior art to capture RNA in sections, followed by reverse transcription to obtain whole transcriptome information
Implementation Method 3
The gap generated between the 3' end of the oligonucleotide sequence fixed on the chip and the 5' end of the random probe can be filled by T4 ligase
Data Source
Figure 1A~1B
Figure 2
Figure 3A~3B
AI summary
Disclose in the present invention are a splint sequence and a use thereof in nucleic acid capture. The first base at the 5' end of the splint sequence is U. During nucleic acid capture, by using the splint sequence provided by the present invention, the effect of effectively reducing the number of adapters generated by the splint sequence in a sequencing library can be achieved, the mapping rate of sequencing data can be improved, and the sequencing costs can be reduced.