Mixed-Enzyme DNA Adapter Ligation for Higher 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 affects the quality of sequencing libraries, and existing methods are not sufficiently effective.
Innovation Solution
A method involving a mixed enzyme reaction system comprising a DNA ligase and an RNA ligase, specifically using T4 RNA ligase, is employed to ligate double-stranded DNA fragments with adapters, enhancing ligation efficiency by up to 85%.
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 T4 RNA ligase in a single mixed enzyme reaction system. The T4 RNA ligase, traditionally used for RNA ligation, is repurposed to work synergistically with DNA ligase for DNA fragment ligation, achieving over 85% efficiency while maintaining operational simplicity
Solution Approach 2:
The patent applies T4 RNA ligase, an enzyme originally designed for RNA ligation, to also facilitate DNA ligation when combined with DNA ligase. This multi-functional application of T4 RNA ligase resolves the contradiction by improving DNA ligation efficiency without requiring a completely new enzyme system
2Productivity
If conventional ligation methods are used, then the process is straightforward, but the yield of effective sequencing library is low
Solution Approach 1:
By merging DNA ligase and T4 RNA ligase activities in one reaction system, the patent simultaneously improves ligation efficiency and sequencing library yield to over 85%, while the mixed enzyme system maintains ease of operation through a single-step protocol
Solution Approach 2:
The mixed enzyme system performs multiple functions self-service: DNA ligase handles primary ligation while T4 RNA ligase enhances the reaction, and T4 polynucleotide kinase prepares DNA ends for ligation, all within the same reaction system without requiring separate processing steps
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 improves the yield and quality of sequencing libraries by achieving high ligation efficiency and reducing adapter residue, making the process more convenient and cost-effective.
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 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 includes ligating a first nucleic acid fragment and a second nucleic acid fragment in a mixed enzyme reaction system including a DNA ligase and an 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.