Nucleic Acid Sequencing via Stabilized Ternary Complexes
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Solution Overview
Problem
Current nucleic acid sequencing methods are inadequate for rapid, accurate, and cost-effective diagnosis in clinical applications, as they often require extensive time and resources, and are prone to errors due to light-based detection methods that can damage nucleic acids.
Innovation Solution
A method involving a primer-template nucleic acid hybrid with a mixture of nucleotides, including reversible terminators and extendable nucleotides, to form stabilized ternary complexes, allowing for the detection and determination of base multiplets without extensive light exposure, thereby reducing sequencing cycles and increasing read length.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If light-based detection methods are used to detect nucleic acid sequences, then detection capability is improved, but nucleic acid damage occurs due to light exposure
Solution Approach 1:
The patent extracts the detection function from light-based methods and implements it through chemical fluorescence signals generated during primer extension. The detection is achieved by monitoring fluorescence intensity changes as nucleotides are incorporated, eliminating the need for separate light-based detection steps that damage nucleic acids.
Solution Approach 2:
The patent replaces the optical detection system with a chemical-based fluorescence detection system. Instead of using light to directly detect and sequence nucleic acids, the system uses fluorescently labeled nucleotides that emit light only when incorporated into the growing primer, substituting mechanical/optical processes with chemical reactions for safer detection.
2Measurement precision
If extensive light exposure is used during sequencing, then detection accuracy is improved, but sequencing cycles increase and read length decreases
Solution Approach 1:
The patent enables continuous sequencing cycles by using reversibly terminated nucleotides that can be incorporated and then chemically deblocked for the next cycle. The fluorescence detection occurs continuously during each extension step without requiring extensive light exposure between cycles, maintaining detection accuracy while enabling rapid sequential cycling and longer read lengths.
Solution Approach 2:
The patent changes the detection parameter from light intensity measurement to fluorescence intensity measurement during chemical reactions. This allows detection to occur during the actual extension process rather than requiring separate exposure steps, improving productivity while maintaining accuracy through real-time monitoring of nucleotide incorporation.
3Reliability
If current sequencing methods are used to ensure accuracy, then diagnostic reliability is improved, but time required and cost increase
Solution Approach 1:
The patent performs preliminary chemical modifications by using reversibly terminated nucleotides with built-in blocking groups that prevent further extension. This preliminary action allows single-base incorporation per cycle with high accuracy, and the blocking groups are later removed chemically to enable continuous cycling, reducing total sequencing time while maintaining diagnostic reliability through controlled, accurate base identification.
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 sequencing speed, accuracy, and cost-effectiveness by reducing nucleic acid exposure to light and chemicals, enabling longer read lengths and rapid identification of nucleic acid sequences.
Implementation Method 1
contacting the extended primer hybrid with a nucleotide cognate for at least one of the different base types and a polymerase to form a stabilized ternary complex that includes the extended primer hybrid, the polymerase and a nucleotide cognate of the next base in the template
Data Source
AI summary
A method of characterizing a nucleic acid that includes steps of (a) contacting a primer-template nucleic acid hybrid with a polymerase and a mixture of nucleotides to produce an extended primer hybrid and to form a stabilized ternary complex including the extended primer hybrid, the polymerase and a nucleotide cognate of the next base in the template, wherein the mixture contains nucleotide cognates of four different base types, wherein the nucleotide cognate of the first base type has a reversible terminator, and wherein nucleotide cognates of the second, third and fourth base types are extendable; (b) detecting the stabilized ternary complex to distinguish the next base from other base types in the template; and (c) determining the presence of a base multiplet in the template nucleic acid, the base multiplet including the first base type followed by the next base.