Polymerase-Binding Molecule Complex for Primer Extension Homogeneity
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Solution Overview
Problem
Nucleic acid polymerases in molecular biology research and diagnostics face issues with mispriming and template or primer degradation, leading to reduced yield and homogeneity of primer extension products, which affects detection sensitivity.
Innovation Solution
The use of a composition comprising a nucleic acid polymerase, a polymerase-binding molecule (PBM) that does not inhibit polymerase activity, and a PBM-binding molecule or complex that together inhibits polymerase activity, allowing for enhanced yield and homogeneity of primer extension products by controlling polymerase activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If polymerase activity is maintained at high levels, then productivity of nucleic acid synthesis is improved, but mispriming and template degradation occur leading to reduced manufacturing precision
Solution Approach 1:
The patent introduces an antibody as an intermediary molecule that specifically binds to the polymerase enzyme. This antibody acts as a mediator that modulates polymerase activity by preventing mispriming and template degradation without completely inhibiting the desired nucleic acid synthesis, thus resolving the contradiction between productivity and manufacturing precision
Solution Approach 2:
The patent employs temperature-dependent parameter changes to control polymerase activity. By maintaining the reaction at temperatures where the antibody-polymerase complex is stable, the system achieves selective inhibition of harmful polymerase activities while preserving productive synthesis, thereby improving both yield and homogeneity
2Manufacturing precision
If polymerase activity is inhibited to prevent mispriming, then manufacturing precision is improved, but yield of primer extension products decreases
Solution Approach 1:
The patent applies partial inhibition of polymerase activity through antibody binding. Rather than completely suppressing polymerase activity, the antibody provides selective partial inhibition that prevents mispriming and template degradation while allowing sufficient productive synthesis to maintain high yield, thus resolving the contradiction between precision and productivity
3Ease of operation
If temperature is maintained for manufacturing and shipping, then ease of operation is improved, but template and primer degradation occur reducing measurement precision
Solution Approach 1:
The patent applies preliminary action by incorporating the antibody into the polymerase preparation before manufacturing and shipping. This pre-formed antibody-polymerase complex provides protective inhibition that prevents template and primer degradation during storage and transport, ensuring that detection sensitivity is maintained when the reagents are later used
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 improves the yield and homogeneity of nucleic acid synthesis products by selectively inhibiting polymerase activity, thereby enhancing detection sensitivity and specificity in molecular biology techniques.
Implementation Method 1
a polymerase-binding molecule (PBM) that binds to the polymerase and does not substantially inhibit the polymerase activity of the polymerase
Implementation Method 2
a PBM-binding molecule or complex of molecules that binds to the PBM such that binding of the PBM and PBM-binding molecule or complex together substantially inhibits the polymerase activity of the polymerase
Data Source
AI summary
The present invention provides multi-component inhibitors of nucleic acid polymerases, methods of making, and methods of using same. One component of the multi-component inhibitor is a molecule that binds to a polymerase (i.e., a polymerase-binding molecule (PBM)), but does not thereby substantially inhibit its polymerase activity. Another component is a molecule or complex of molecules that binds to a PBM (i.e., a PBM-binding molecule). The combination of the PBM and PBM-binding molecule/complex substantially inhibits polymerase activity. The disclosed multi-component inhibitors are useful for DNA sequencing, nucleic acid amplification, cloning and synthesis, and the like.