Mixed-Enzyme DNA Fragment Ligation for Sequencing Library Yield
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Solution Overview
Problem
The efficiency of nucleic acid fragment ligation, particularly the ligation of adapters to DNA molecules, significantly impacts the quality of sequencing libraries, with existing methods often being inefficient and cumbersome.
Innovation Solution
A method involving a mixed enzyme reaction system comprising DNA ligase and RNA ligase, specifically using T4 RNA ligase, enhances the ligation efficiency of double-stranded DNA fragments to adapters, with optional use of T4 polynucleotide kinase for phosphorylation, achieving high yields and simplified operations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If only DNA ligase is used for double-stranded DNA fragment ligation, then the reaction system is simple, but the ligation efficiency is low (38.14-77.5%)
Solution Approach 1:
The patent combines DNA ligase and RNA ligase in a single mixed enzyme reaction system. This merging of two different enzyme systems allows the RNA ligase to enhance DNA fragment ligation efficiency while maintaining a unified reaction protocol, resolving the contradiction between improving ligation efficiency and maintaining system simplicity.
Solution Approach 2:
The mixed enzyme system exhibits multi-functionality where RNA ligase, traditionally used for RNA ligation, also enhances DNA fragment ligation. This universal application of RNA ligase across different nucleic acid types improves DNA ligation efficiency without requiring a completely new enzyme system, thus balancing efficiency improvement with system complexity.
2Productivity
If a mixed enzyme reaction system with DNA ligase and RNA ligase is used, then ligation efficiency increases (over 85%), but the reaction system becomes more complex
Solution Approach 1:
By merging DNA ligase and RNA ligase into a single mixed enzyme reaction system, the patent achieves high ligation efficiency (>85%) while maintaining operational simplicity. The combined system allows both enzymes to work simultaneously in one reaction mixture, eliminating the need for separate ligation steps and simplifying the overall操作流程 despite the increased enzymatic complexity.
3Productivity
If traditional ligation methods are used, then the procedure is straightforward, but the yield of effective sequencing libraries is low
Solution Approach 1:
The mixed enzyme system merges the functions of DNA ligase and RNA ligase to dramatically improve library yield. This combination eliminates the need for extensive parameter optimization and multiple trial experiments, as the synergistic effect of the two enzymes provides robust ligation performance directly, reducing both time loss and improving productivity.
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 achieves ligation efficiencies over 85% and improves the yield of effective sequencing libraries, offering convenience and cost-effectiveness.
Implementation Method 1
ligating a first nucleic acid fragment and a second nucleic acid fragment in a mixed enzyme reaction system comprising a DNA ligase and an RNA ligase
Implementation Method 2
the ligation efficiency between the adapter and nucleic acids directly affects the quality of the constructed sequencing library
Implementation Method 3
the phosphorylation is performed using a polynucleotide kinase
Data Source
AI summary
Disclosed are a method for ligating nucleic acid fragments, a method for constructing a sequencing library, and the use thereof. The method for ligating nucleic acid fragments comprises ligating a first nucleic acid fragment and a second nucleic acid fragment in a mixed enzyme reaction system comprising a DNA ligase and a RNA ligase, wherein the first nucleic acid fragment is double-stranded DNA. The ligation efficiency of the method for ligating nucleic acid fragments can reach 85% or more, and by using this ligation method to construct a sequencing library, the yield of the effective library is significantly improved.