Evaluating DNA Adapter Ligation Efficiency via Electrophoresis
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Solution Overview
Problem
Current methods for evaluating the efficiency of adapter ligation in DNA sequencing, particularly for scarce DNA samples like those used in liquid biopsies, lack a convenient and accurate means to optimize reaction conditions, leading to incomplete adapter ligation and reduced sequencing precision.
Innovation Solution
A method using model DNA fragments and adapters to evaluate ligation efficiency through electrophoresis, allowing for the optimization of reaction conditions by analyzing the mobility of ligation molecules, ensuring complete adapter binding for improved sequencing accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional ligation methods with recommended conditions are used, then the process is simple to operate, but the ligation efficiency cannot be evaluated leading to incomplete adapter binding
Solution Approach 1:
The patent introduces an evaluation method using electrophoresis as an intermediary technique to assess ligation efficiency. By running the ligation reaction products through electrophoresis, the method visualizes adapter-DNA complexes and quantifies ligation efficiency based on band intensity ratios, providing a measurable indicator without fundamentally changing the ligation process itself
Solution Approach 2:
The patent replaces subjective visual assessment of ligation completeness with an objective electrophoresis-based measurement system. Instead of relying on mechanical mixing and visual inspection, the method uses electrophoretic separation and densitometric analysis to precisely quantify ligation efficiency, substituting mechanical simplicity with analytical precision
2Reliability
If Y-type adapters are ligated to both ends of double-stranded DNA, then sequencing accuracy is improved through complete ligation, but the reaction conditions are difficult to optimize without evaluation methods
Solution Approach 1:
The patent implements a feedback mechanism where electrophoresis results provide quantitative information about ligation efficiency. This feedback loop allows researchers to adjust ligation conditions (enzyme amount, reaction time, temperature) based on measured efficiency values, systematically optimizing the reaction to achieve near-complete adapter binding and thereby maximizing sequencing accuracy
Solution Approach 2:
The patent enables systematic variation of ligation parameters (enzyme concentration, reaction time, temperature, adapter-to-DNA ratio) with electrophoresis-based evaluation. By changing these parameters and measuring their effect on ligation efficiency, the optimal conditions for complete adapter binding can be identified, ensuring high reliability in sequencing applications
3Ease of operation
If scarce DNA samples like cfDNA are used in liquid biopsy, then minimally invasive diagnosis is achieved, but sequencing precision is reduced due to limited sample amount
Solution Approach 1:
The patent applies partial action by using electrophoresis to evaluate only the critical ligation step rather than sequencing the entire scarce DNA sample. By measuring ligation efficiency on a subset of the DNA and optimizing based on this partial information, the method ensures high precision in the adapter binding step without consuming excessive amounts of the limited cfDNA sample
Solution Approach 2:
The patent performs preliminary evaluation of ligation efficiency using electrophoresis before proceeding to full sequencing. This preliminary assessment allows optimization of adapter binding completeness on scarce DNA samples, ensuring that the subsequent sequencing step operates on maximally ligated products, thereby preserving measurement precision despite limited sample availability
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 enables the convenient and accurate evaluation of adapter ligation efficiency, optimizing reaction conditions to achieve high precision and completeness in adapter binding, enhancing the efficiency and accuracy of DNA sequencing, especially in liquid biopsies and general DNA sequencing.
Implementation Method 1
the efficiency of the reaction is evaluated by electrophoresing a reaction mixture containing ligation molecules
Data Source
AI summary
The purpose of the present invention is to provide a method for conveniently and accurately evaluating the ligation efficiency in the DNA sequencing process in order to optimize the condition of ligating Y-type adapters to both ends of a double-stranded DNA fragment. The present invention relates to a method for evaluating the efficiency of ligation reaction through which Y-type adapters are ligated to both ends of DNA to be analyzed, in the sequencing process of DNA to be analyzed using the Y-type adapter, wherein the efficiency of reaction is evaluated by electrophoresing a reaction mixture containing ligation molecules, between the DNA and the Y-type adapters, produced by the ligation reaction under a specified condition, and analyzing a band separated on the basis of the number of adapters ligated to the DNA.