RNA and DNA Library Construction with Enzymatic Linker Cleanup
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Solution Overview
Problem
Existing methods for constructing sequencing libraries of small RNAs or RNA fragments face challenges in efficiently removing linker self-linking products, especially in low-abundance samples, leading to hindered PCR amplification and incomplete library construction, while integrated DNA and RNA sequencing methods struggle with sample heterogeneity and require separate library preparations.
Innovation Solution
A method involving the use of endonucleases, specifically designed linkers with partial recognition sites, and enzymes like RNase H to efficiently cleave self-linking products, followed by PCR amplification to construct sequencing libraries for RNA and DNA molecules, allowing simultaneous sequencing of mixed RNA and DNA samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If linker ligation is performed to construct sequencing libraries of small RNAs or RNA fragments, then library construction is achieved, but linker self-linking products are generated that hinder PCR amplification
Solution Approach 1:
The patent extracts and removes the harmful linker self-linking products from the reaction mixture using specific enzymes (exonucleases to degrade free linkers and endonucleases to cleave self-linking products), thereby separating the desired RNA-linker complexes from the contaminating linker-only products, enabling successful PCR amplification and sequencing library construction
Solution Approach 2:
The patent introduces enzymatic intermediaries (phosphatases, kinases, exonucleases, and endonucleases) that mediate the selective modification and degradation of linker molecules. These enzymes act as intermediaries to distinguish between RNA-linked and unlinked linkers, enabling selective removal of self-linking products while preserving the desired library molecules
2Object-generated harmful factors
If existing removal methods like CleanTag technology or Cas9 enzyme are used, then linker self-linking products are removed, but the process becomes costly and operationally complicated
Solution Approach 1:
The patent employs commercially available, cost-effective enzymatic reagents (phosphatases, kinases, exonucleases, and endonucleases) that can be purchased off-the-shelf rather than requiring expensive specialized systems like CleanTag technology or Cas9. These enzymes provide a simple, disposable solution that eliminates the need for complex experimental setups and specialized equipment
Solution Approach 2:
The patent changes the chemical parameters of the linker molecules by introducing specific modifications (5'-phosphate, 3'-hydroxyl groups) that enable selective enzymatic recognition and degradation. By controlling the phosphorylation state and using enzymes with specific substrate requirements, the method achieves selective removal of self-linking products through parameter-based differentiation rather than complex procedural steps
3Adaptability or versatility
If separate library preparations are performed for DNA and RNA sequencing, then comprehensive genomic and transcriptomic analysis is achieved, but the process requires multiple separate procedures
Solution Approach 1:
The patent merges the DNA and RNA library preparation procedures into a single unified workflow. By using the same linker molecules and enzymatic treatment steps for both DNA and RNA samples, the method enables simultaneous construction of both libraries from the same starting materials, eliminating the need for separate preparation protocols while maintaining the ability to perform comprehensive genomic and transcriptomic analysis
Solution Approach 2:
The patent creates universal linker molecules and enzymatic reagents that can process both DNA and RNA samples through the same protocol. The linkers are designed to work with both nucleic acid types, and the enzymatic treatments (phosphatase, kinase, exonuclease, endonuclease) are applicable to both DNA and RNA libraries, providing a multi-functional solution that simplifies the overall workflow
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
The method effectively removes up to 99.9% of self-linking products, enabling comprehensive genomic and transcriptomic analysis of low-abundance RNA samples and integrated sequencing of DNA and RNA in mixed samples, improving accuracy and efficiency.
Implementation Method 1
both the 5' end linker and the 3' end linker contain a partial sequence of a recognition site of the DNA endonuclease, and when a self-linking product of the 5' end linker and the 3' end linker is formed, a complete sequence of the recognition site is formed at the junction
Implementation Method 2
treating the extension product using a DNA endonuclease and an enzyme capable of specifically hydrolyzing an RNA in a DNA-RNA heterozygous chain
Data Source
AI summary
The present invention relates to a method and kit for removing linker self-linking products during sequencing library construction and for constructing a sequencing library. In particular, provided is a method and kit for the selective cleavage of DNA double strands and DNA single strands, a method and kit for selective cleavage by using DNA-RNA-DNA:cDNA heterozygous chains and DNA:cDNA double strands, and a method and kit for DNA and RNA library co-construction.