Modified Nucleobase Sequencing via Reversible Covalent Ligation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current DNA sequencing and SNP analysis methods rely heavily on enzymes and complex triphosphates, which are costly and prone to errors, limiting read lengths and accuracy.
Innovation Solution
Development of modified nucleobase compounds with reversible covalent reactions and detectable tags, allowing for chemical-based DNA sequencing and SNP characterization without the need for enzymes, using aldehyde or ketone moieties and fluorescent or mass-tag labels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If enzyme-based methods (Sanger sequencing, pyrosequencing, restriction enzyme mediated cleavage) are used for DNA sequencing and SNP analysis, then the sequencing process can be performed with established biological mechanisms, but the cost increases, errors are more prone, and read lengths are limited
Solution Approach 1:
The patent replaces the biological enzymatic system with a chemical ligation system. Specifically, it uses DNA ligase to join a primer to a template strand containing a SNP, followed by chemical cleavage and ligation steps that do not require additional enzymes. This substitution of biological mechanisms with chemical ones reduces complexity and cost while maintaining or improving accuracy.
Solution Approach 2:
The patent extracts and isolates the specific SNP detection step from the complex enzymatic sequencing process. By focusing on a targeted ligation-based approach that only requires DNA ligase and specific oligonucleotides, it removes the need for multiple enzyme types and complex triphosphate reagents, thereby reducing overall system complexity while improving reliability.
2Ease of manufacture
If chemical-based sequencing methods are used, then costs are reduced and read lengths are improved, but the methods require new chemical reactions and reagents
Solution Approach 1:
The patent segments the sequencing process into distinct chemical steps: initial ligation of primer to template, cleavage of the ligated product, and subsequent ligation reactions. Each step uses simple, well-established chemical reactions (ligase-mediated joining and base-specific cleavage) rather than requiring new complex reagents, making the method both cost-effective and adaptable to existing laboratory infrastructure.
3Productivity
If multiple enzymes and complex triphosphates are used in sequencing reactions, then the sequencing process can proceed through multiple steps, but the number of reagents increases and errors are more prone
Solution Approach 1:
The patent employs DNA ligase as a universal enzyme that performs multiple functions: joining the primer to the template strand, and subsequently joining labeled oligonucleotides in a second ligation step. This multi-functionality eliminates the need for multiple different enzymes, reducing the quantity of reagents required while maintaining high productivity through sequential chemical reactions.
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
Enables efficient and accurate DNA sequencing and SNP analysis with improved read lengths and reduced costs, as no enzymes are required, and the use of labeled PNA monomers simplifies the process.
Implementation Method 1
a moiety capable of reversible covalent reactions selected from the group consisting of (i) aldehyde and (ii) ketone
Implementation Method 2
fluorescent or mass-tag labels
Data Source
Figure 1
Figure 2
Figure 3
AI summary
The present invention provides modified nucleobase compounds, modified nucleic acid mimetic compounds and various uses thereof. In addition, the invention provides methods for nucleobase characterisation, SNP characterisation and nucleic acid sequencing.