Sequencing Library Prep Using DNA-Binding Dyes to Suppress mtDNA
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Enzymatic systems in molecular biology and genomics suffer from off-target effects, leading to difficulty in analyzing results due to poor substrate discrimination, particularly between nucleic acids like mitochondrial DNA (mtDNA) and nuclear DNA.
Innovation Solution
The use of DNA-binding molecules, such as Hoechst dye, SYBR Gold, Sytox Orange, and Pico Green, to preferentially bind and inhibit secondary nucleic acids, such as mtDNA, thereby reducing or eliminating their sequencing reads during enzymatic reactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If enzymatic reactions are performed on nucleic acid samples, then sequencing reads are generated, but off-target effects occur causing poor substrate discrimination between mitochondrial DNA and nuclear DNA
Solution Approach 1:
The patent introduces an intermediary DNA-binding molecule that selectively binds to mitochondrial DNA, acting as a mediator to prevent the enzymatic system from directly interacting with the off-target substrate. This intermediary blocks the enzyme's access to mitochondrial DNA while allowing it to process nuclear DNA, thereby resolving the substrate discrimination problem without modifying the enzymatic system itself
Solution Approach 2:
The patent changes the chemical parameters of the reaction system by introducing DNA-binding molecules with specific binding affinities. By selecting molecules like Hoechst dye, SYBR Gold, or Sytox Orange that have preferential binding to mitochondrial DNA, the system alters the effective concentration and accessibility of different nucleic acid substrates, enabling the enzyme to distinguish between mitochondrial and nuclear DNA based on differential binding parameters
2Reliability
If DNA-binding molecules are used to preferentially bind secondary nucleic acids, then off-target effects are reduced, but the complexity of the system increases
Solution Approach 1:
The patent employs inexpensive, readily available DNA-binding dyes and molecules that can be added to the reaction mixture in a single step. These molecules serve their purpose of blocking off-target substrates during the enzymatic reaction and then can be discarded or diluted out,无需 complex removal or recovery processes. This approach maintains high reliability while minimizing system complexity
Solution Approach 2:
The patent utilizes DNA-binding molecules that have the dual function of both staining/visualizing DNA and simultaneously blocking off-target enzymatic activity. Molecules like SYBR Gold and Hoechst dye serve multiple purposes: they bind to mitochondrial DNA to prevent enzymatic processing while also potentially serving as fluorescent markers for later detection, thereby reducing overall system complexity through multi-functionality
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 enhances the specificity of enzymatic reactions by selectively targeting primary nucleic acids of interest, reducing off-target effects and improving the accuracy of sequencing results.
Implementation Method 1
contacting the sample with a DNA-binding molecule that preferentially binds the secondary nucleic acids
Data Source
AI summary
Embodiments of systems, methods, and compositions provided herein relate to assays for selectively controlling enzymatic reactions. Some embodiments relate to methods of inhibiting, reducing, or eliminating secondary DNA (such as mitochondrial DNA) sequencing reads from open chromatic sequencing, whole genome sequencing, or targeted sequencing.