Linker Element for Directional Sequencing Library Construction
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Solution Overview
Problem
The existing methods for constructing sequencing libraries require multiple steps and expensive reagents, leading to high costs and inefficiencies due to the need for multiple linker ligation steps and purification processes.
Innovation Solution
A novel linker ligation method using a unique linker configuration and single-strand replacement, reducing the linker ligation process to four steps and eliminating the need for intermediate purification, while ensuring directional ligation and preventing linker interconnection, combined with nucleic acid probe capture technology for target genomic region sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If multiple linker ligation steps are performed to add linkers to both ends of DNA fragments, then directional ligation is achieved, but the construction time and cost increase significantly
Solution Approach 1:
The patent combines the ligation of both linkers into a single simultaneous reaction step. The linker molecule contains two different linkers (Linker 1 and Linker 2) connected by a spacer sequence, allowing both linkers to be ligated to opposite ends of DNA fragments in one reaction rather than requiring separate sequential ligation steps.
Solution Approach 2:
The linker molecule serves multiple functions simultaneously: it acts as both Linker 1 and Linker 2, provides the spacer sequence for directional orientation, and includes recognition sequences for both R1 and R2 nucleic acid probes. This multi-functional design eliminates the need for separate linker molecules and multiple ligation reactions.
2Reliability
If multiple purification steps are performed between ligation reactions, then reaction completeness is improved, but the overall process cost and complexity increase
Solution Approach 1:
The patent merges multiple ligation reactions into a single reaction system and eliminates intermediate purification steps. The simultaneous ligation of both linkers in one reaction step, followed by a single amplification reaction, replaces the conventional multi-step process with intermediate purifications, thereby reducing process complexity while maintaining reaction completeness.
3Manufacturing precision
If sticky ends are used to link linkers, then directional ligation is achieved, but fragment interconnection becomes difficult to avoid
Solution Approach 1:
The patent introduces a spacer sequence as an intermediary element between Linker 1 and Linker 2 in the linker molecule. This spacer sequence prevents direct interaction between the linkers that would cause fragment interconnection, while still allowing the linkers to be ligated directionally to opposite ends of the DNA fragment through complementary base pairing.
4Quantity of substance
If conventional multi-step linker ligation is performed, then both ends of DNA fragments can be ligated, but reagent cost increases due to multiple extension reactions
Solution Approach 1:
The patent combines the ligation of both linkers into a single reaction step using one ligase enzyme, rather than requiring separate ligation reactions for each linker. This is followed by a single amplification reaction using one polymerase enzyme, significantly reducing the consumption of expensive enzymes and other reagents compared to the conventional multi-step process.
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 reduces the time and cost of linker ligation, prevents linker interconnection, and enables efficient construction of a sequencing library, allowing for cost-effective and accurate sequencing of target genomic regions.
Implementation Method 1
the linker A is generated from the complementary pairing of a long strand of nucleic acid and a short strand of nucleic acid
Implementation Method 2
the application derived from the second-generation sequencing combined with microarray technology—target sequence capture sequencing technology can use a large number of oligonucleotide probes to complementarily bind to specific regions in the genome
Data Source
AI summary
Provided is a linker element and a method of using the linker element to construct a sequencing library, wherein the linker element consists of a linker A and a linker B, the linker A is obtained through the complementary pairing of a long nucleic acid strand and a short nucleic acid strand, the 5′ end of the long strand has a phosphoric acid modification, and the 3′ end of the short strand has an enclosed modification, with enzyme sites in the short strand; and the linker B is a nucleic acid single strand, and the 3′ end thereof can be in a complementary pairing with the 5′ end of the long strand of the linker A. Using the linker element of the present invention for constructing a sequencing library ensures the linking directionality of the linkers while solving the problems of fragment interlinking, linker self-linking and low linking efficiency, and reducing the purification reaction between steps, shortening the linking time and reducing costs.