Li+-Stabilized Ternary Complexes for Low-Background Sequencing
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Solution Overview
Problem
Existing Sequencing By Binding™ (SBB™) technology faces challenges in maintaining detectable levels of ternary complexes while minimizing the concentration of labeled background components, which cause background signal interference.
Innovation Solution
The use of lithium (Li+), betaine, and/or inhibitory metal ions such as Ca2+, along with polyethylenimine (PEI), to stabilize ternary complexes and reduce background noise by replacing excess labeled components in the reaction mixture.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If non-bound labeled nucleotide is maintained in the reaction mixture to preserve ternary complex equilibrium, then the stability of ternary complexes is improved, but background signal increases and obscures detection
Solution Approach 1:
The patent extracts and removes non-bound labeled nucleotides from the reaction mixture after ternary complex formation, while maintaining complex stability through alternative means (labeled polymerase, chelating agents, temperature control). This separation eliminates background signal from free nucleotides while preserving the detectable ternary complexes.
Solution Approach 2:
The patent introduces chelating agents (e.g., EDTA, EGTA) as intermediaries that bind metal ions required for polymerase activity, thereby inhibiting extension of bound nucleotides while allowing ternary complex formation and detection. This mediator enables differentiation between bound and unbound states without requiring labeled nucleotides in excess.
2Measurement precision
If labeled polymerase is used instead of labeled nucleotides to detect ternary complexes, then background signal from free nucleotides is reduced, but the complexity of the system increases
Solution Approach 1:
The patent employs polymerase molecules that serve multiple functions: they catalyze nucleotide incorporation during complex formation, provide the detection label (fluorophore, quantum dot, or other detectable moiety), and enable signal amplification through enzymatic activity. This multi-functionality consolidates detection capabilities into a single reagent type.
3Quantity of substance
If excess polymerase and nucleotide are present in the reaction mixture, then ternary complex formation is enhanced, but background interference from unbound components increases
Solution Approach 1:
The patent performs preliminary formation of ternary complexes under conditions that favor binding (excess polymerase and nucleotide, optimal temperature and pH), then subsequently removes unbound components through washing or chelation before detection. This two-stage approach ensures maximum complex formation while eliminating background interference.
Solution Approach 2:
The patent changes reaction parameters (temperature, pH, ionic strength, presence of chelating agents) to control the equilibrium between bound and unbound states. By adjusting these parameters, the system maximizes ternary complex formation while minimizing the concentration of free, interfering components.
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 stability of ternary complexes, allowing for clearer detection with reduced background interference, thereby improving the accuracy and efficiency of nucleic acid sequencing.
Implementation Method 1
the fluid containing a ternary complex and Li+, wherein the ternary complex includes a primed template nucleic acid, a polymerase, and a nucleotide cognate
Implementation Method 2
betaine and/or a metal ion that inhibits polymerase catalysis such as Ca2+
Data Source
AI summary
Methods, compositions, kits and apparatuses that include a fluid, the fluid containing a ternary complex and Li+, wherein the ternary complex includes a primed template nucleic acid, a polymerase, and a nucleotide cognate for the next correct base for the primed template nucleic acid molecule. As an alternative or addition to Li+, the fluid can contain betaine or a metal ion that inhibits polymerase catalysis such as Ca2+. In addition to Li+, the fluid can contain polyethylenimine (PEI) with or without betaine.