Analyte Detection Assay Using Polymerase Segmentation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for detecting analytes, such as immuno-PCR assays, face challenges with high background signals and low sensitivity due to non-specific binding of DNA and antibodies, limiting the ability to detect small quantities of analytes effectively.
Innovation Solution
The use of a binding molecule coupled to a first polymerase that amplifies a modified nucleic acid template, preventing non-specific amplification by a second polymerase, which can only amplify an unmodified copy, thereby reducing background noise and increasing sensitivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If DNA is linked to antibodies via a linker with bisspecific affinity, then the formation of specific antigen-antibody-DNA conjugate is achieved, but non-specific binding of unbound DNA occurs giving rise to high background signal
Solution Approach 1:
The patent extracts the harmful unbound DNA from the system by implementing a wash step that removes unbound DNA before the detection phase. This allows only specifically bound DNA-antibody conjugates to remain in the assay, eliminating non-specific background binding while preserving specific antigen detection capability
Solution Approach 2:
The patent performs preliminary washing steps before the detection phase to remove unbound DNA from the system. This preliminary action prevents unbound DNA from contributing to background signals during subsequent detection steps, thereby improving measurement precision
2Object-generated harmful factors
If blocking solutions containing inert proteins are added to reduce unspecific binding, then unspecific binding is reduced, but background signal is not eliminated resulting in lower sensitivity
Solution Approach 1:
Instead of relying on blocking solutions that partially reduce non-specific binding, the patent extracts the harmful unbound DNA through washing steps. This complete removal of unbound DNA eliminates background signal entirely, thereby achieving both reduced non-specific binding and maintained sensitivity
Solution Approach 2:
The patent uses PCR amplification to create multiple copies of the bound DNA template. This copying process amplifies the specific signal from bound DNA while the wash steps ensure no amplified background signal is present, thereby improving sensitivity without compromising specificity
3Device complexity
If a single polymerase is used to amplify the template, then the amplification process is simple, but sensitivity is limited and background interference occurs
Solution Approach 1:
The patent segments the polymerase function into two distinct polymerases: a first polymerase that synthesizes the initial template copy and a second polymerase that performs the amplification. This segmentation allows each polymerase to have specialized functions, with the second polymerase optimized for high-sensitivity amplification while the first handles template synthesis, thereby improving overall sensitivity
Solution Approach 2:
The patent introduces an intermediate unmodified DNA template as a mediator between the first polymerase and the second polymerase. The first polymerase creates this intermediate unmodified template from the modified template, which then serves as the substrate for the second polymerase's amplification reaction. This intermediary step enables the system to achieve high sensitivity while maintaining process simplicity
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 sensitivity and reproducibility by minimizing background signals and improving the detection of analytes, allowing for more accurate quantification of analytes in solutions.
Implementation Method 1
the polymerase provides a means to generate a template for a nucleic acid amplification reaction
Implementation Method 2
analyte-specific binding molecules having a binding moiety coupled to a polymerase or a portion thereof. Upon binding of the analyte to the binding moiety
Data Source
AI summary
The present invention provides methods, kits and compositions for the detection of an analyte. In the methods of the invention, a binding molecule coupled to a first polymerase is incubated with a modified polynucleotide template to form a copy of the modified polynucleotide template without any modified nucleotides. The unmodified copy is detected in a second amplification/primer extension reaction using a second polymerase that is unable to amplify the modified polynucleotide template. Detection of the unmodified copy is indicative of the presence and/or amount of the analyte in the sample. In place of the binding molecule coupled to a first polymerase, a pair of analyte-specific probes can also be used. The first analyte specific probe comprises a first binding moiety and a first portion of a first polymerase and the second analyte specific probe comprises a second binding moiety and a second portion of the first polymerase. When the binding moieties are bound to the analyte the first and second portions of the polymerase interact to form a functional polymerase complex that is used to form a copy of the modified polynucleotide template without any modified nucleotides.