Reversible Nucleotide Terminators for High-Throughput DNA Sequencing
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Solution Overview
Problem
Current DNA sequencing technologies are expensive, labor-intensive, and time-consuming, making them unsuitable for broad application in human sequence variation studies, particularly in identifying rare and dispersed genetic variants associated with diseases.
Innovation Solution
Development of novel compounds with photocleavable, enzymatically cleavable, or non-photocleavable groups for use in high-throughput DNA sequencing methods, including nucleotides and nucleosides that facilitate efficient sequencing by terminating DNA synthesis and allowing for rapid detection and identification of genetic variations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional Sanger sequencing or early next-generation sequencing methods are used, then DNA sequencing can be performed, but the process is expensive, labor-intensive, and time-consuming
Solution Approach 1:
The sequencing process is divided into discrete cycles, each incorporating a single nucleotide type with a reversible terminator. This segmentation allows parallel processing of multiple sequencing reactions and enables automated high-throughput sequencing by breaking down the complex sequencing task into manageable, repeatable units that can be performed simultaneously across millions of DNA molecules.
Solution Approach 2:
The patent employs periodic action through cyclic reversible termination, where sequencing occurs in repeated cycles of nucleotide incorporation, imaging, and terminator removal. Each cycle adds one nucleotide and is followed by a cleavage step that restores the 3'-OH group, enabling the next cycle to proceed. This periodic pattern allows continuous sequencing without manual intervention between steps, dramatically increasing throughput while reducing overall sequencing time.
2Measurement precision
If traditional sequencing methods are used, then genetic variants can be identified, but the process requires significant manual labor and complex procedures
Solution Approach 1:
The system employs self-service through automated nucleotide incorporation and detection. DNA polymerase automatically selects and incorporates the correct nucleotide based on template complementarity, and fluorescent reporters automatically signal the incorporated base type. The reversible terminators self-cleave under specific conditions to restore functionality for the next cycle, eliminating the need for manual intervention in each sequencing step and simplifying operation while maintaining high precision.
Solution Approach 2:
The patent uses color changes through fluorescent reporters attached to each nucleotide type. Each nucleotide (A, C, G, T) is labeled with a distinct fluorophore that emits a characteristic color when excited. This optical signal allows automated detection and identification of incorporated nucleotides, replacing manual analysis with objective, high-precision optical measurement that can be processed by computer algorithms, thereby improving both accuracy and ease of operation.
3Loss of information
If current sequencing technologies are applied to whole genome sequencing, then complete genetic information can be obtained, but the cost and time requirements make broad application impractical
Solution Approach 1:
The patent merges millions of individual sequencing reactions into a single pooled sample. All DNA molecules are prepared in parallel, then combined and subjected to the same sequencing cycles. This merging allows the cost and time of reagents and instrumentation to be amortized across a vast number of samples simultaneously, reducing the cost per sample from thousands to potentially hundreds or tens of dollars while maintaining complete genomic information through the collective data from all pooled molecules.
Solution Approach 2:
The sequencing system achieves universality through the use of universal primers and reagents that can sequence any DNA template. The same DNA polymerase, nucleotides, and detection system work for all samples in the pool, regardless of their specific genetic content. This multi-functionality eliminates the need for sample-specific optimization and allows a single sequencing run to generate complete genomic data for numerous individuals, dramatically reducing per-sample costs while maintaining information completeness.
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
These compounds enable efficient and accurate sequencing of genomic information, reducing costs and time by improving the incorporation efficiency and deprotection properties, allowing for the identification of rare genetic variants and whole genome sequencing.
Implementation Method 1
photocleavable groups and methods for their use in a number of DNA sequencing methods
Data Source
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AI summary
The present invention relates generally to labeled and unlabled cleavable terminating groups and methods for DNA sequencing and other types of DNA analysis. More particularly, the invention relates in part to nucleotides and nucleosides with chemically cleavable, photocleavable, enzymatically cleavable, or non-photocleavable groups and methods for their use in DNA sequencing and its application in biomedical research.