Siderophore Chelators Reduce DNA Oxidation in Sequencing
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Solution Overview
Problem
Current sequencing-related sample preparation methods using EDTA lead to severe and irreversible oxidation of DNA, causing local point mutations and erroneous polymorphism calls, especially in cancer-related deep sequencing applications where low-frequency polymorphisms are critical.
Innovation Solution
The use of siderophores, such as desferrioxamine B mesylate salt, in nucleic acid preparation reagents replaces EDTA, reducing oxidative damage by chelating Fe(III) and minimizing the generation of reactive oxygen species, thereby enhancing the quality of sequencing reactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If EDTA is used as a chelator in sequencing sample preparation, then divalent cations are removed effectively, but severe and irreversible oxidation of DNA occurs due to Fe2+(EDTA) complex formation
Solution Approach 1:
The patent changes the chemical parameter of the chelating agent from EDTA to a siderophore-based chelator. This parameter change fundamentally alters the interaction with iron ions, preventing the formation of the Fe2+(EDTA) complex that generates reactive oxygen species, thereby eliminating oxidative damage while maintaining cation removal effectiveness
Solution Approach 2:
The patent converts the potentially harmful interaction between chelators and iron ions into a beneficial outcome. By using siderophores that specifically bind Fe(III) without generating harmful Fe2+ complexes, the system transforms what could be a source of oxidative stress into a protective mechanism that prevents DNA damage while maintaining chelation function
2Reliability
If EDTA is used to chelate divalent cations, then the chelation function is achieved, but point mutations occur in the DNA sample
Solution Approach 1:
The patent changes the chemical identity of the chelator from EDTA to siderophores, which alters the biochemical interaction profile. This parameter change prevents the formation of Fe2+ complexes that cause point mutations, thereby improving both sequencing accuracy and polymorphism call accuracy without sacrificing cation chelation effectiveness
3Reliability
If EDTA is used in sample preparation, then cation removal is effective, but Q score of sequencing reactions decreases
Solution Approach 1:
The patent changes the chelator parameter from EDTA to siderophore-based compounds, which eliminates the generation of reactive oxygen species that degrade DNA quality. This parameter improvement directly increases the Q score of sequencing reactions by maintaining higher DNA integrity throughout the sample preparation process
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 increases the Q score of sequencing reactions, reducing point mutations and improving the accuracy of deep sequencing applications by maintaining DNA integrity, particularly in cancer-related deep sequencing, with Q scores greater than 34, 38, or 42.
Implementation Method 1
siderophore chelators...chelating Fe(III) and minimizing the generation of reactive oxygen species
Implementation Method 2
severe and irreversible oxidation of the DNA especially in the presence of contaminating iron, due to the generation of an Fe2+(EDTA) complex which promotes damaging free-radical chemistry
Data Source
AI summary
Embodiments disclosed herein provide reagents and kits for nucleic acid preparation comprising a siderophore. Embodiments disclosed herein provide methods for preparing a nucleic acid library, which comprise: providing a plurality of nucleic acid molecules from a sample; and manipulating the plurality of nucleic acid molecules in a reagent for nucleic acid preparation comprising a siderophore. Further, embodiments disclosed herein provide methods for reducing oxidative damage to a nucleic acid molecule or increasing the Q (phred) score of a sequencing reaction, which methods comprise preparing the nucleic acid molecule in the absence of EDTA.