Nucleic Acid Tag for Sensitive Protein Detection via qPCR
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Solution Overview
Problem
Traditional methods for protein detection, such as gel electrophoresis and ELISA-based assays, are cumbersome, lack high-throughput capability, and suffer from limitations in detection sensitivity and specificity.
Innovation Solution
Development of nucleic acid tags with reporter functions that can link to proteins of interest, enabling sensitive and selective detection using quantitative PCR, allowing for visualization, quantification, and purification of proteins through radiolabeling, fluorescent labeling, or biotinylation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional methods such as gel electrophoresis and ELISA are used for protein detection, then the detection can be performed with conventional reagents and equipment, but the methods are cumbersome, lack high-throughput capability, and have limited detection sensitivity and specificity
Solution Approach 1:
The patent introduces a nucleic acid tag as an intermediary molecule that bridges the protein of interest and the detection system. The tag comprises a protein-binding domain that specifically binds to the target protein and a reporter sequence that can be detected by quantitative PCR. This intermediary approach enables highly sensitive and specific detection while simplifying the overall procedure and enabling high-throughput analysis, as the nucleic acid tag can be amplified and detected using standardized qPCR workflows.
2Quantity of substance
If traditional protein detection methods are used, then no special tagging is required, but the detection requires expensive assay components and purification steps
Solution Approach 1:
The nucleic acid tag is designed to be self-amplifying through quantitative PCR. The reporter sequence within the tag serves as a template for exponential amplification, generating sufficient signal from minute amounts of target protein without requiring expensive assay components or complex purification steps. The tag essentially performs its own detection function through the amplification of its nucleic acid sequence.
Solution Approach 2:
The patent utilizes the ability to create multiple copies of the nucleic acid tag sequence through PCR amplification. A single protein-bound tag molecule generates millions of copies of its nucleic acid sequence, exponentially amplifying the detection signal. This copying mechanism enables detection of extremely low abundances of target protein while eliminating the need for expensive reagents or purification steps.
3Measurement precision
If nucleic acid tags with reporter functions are used, then high sensitivity and specificity detection is achieved, but the system requires linking nucleic acid sequences to proteins
Solution Approach 1:
The nucleic acid tag is pre-designed and synthesized with both the protein-binding domain and the reporter sequence already linked together. This preliminary construction of the complete tag molecule eliminates the need for complex linking procedures during the actual detection process. The tag can be introduced directly to the sample, and the protein-binding domain automatically anchors it to the target protein while the reporter sequence remains available for qPCR detection.
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
Enables the detection of small amounts of proteins with high sensitivity and specificity, reducing the need for expensive assay components and eliminating the need for purification steps, while allowing for precise detection in complex mixtures.
Implementation Method 1
a second nucleic acid sequence that binds the nucleic acid-interacting motif
Implementation Method 2
a first nucleic acid sequence that is a PCR amplification sequence (an amplicon) recognizable by a PCR probe
Implementation Method 3
fluorescent labeling, or biotinylation
Implementation Method 4
radiolabeling, fluorescent labeling, or biotinylation
Data Source
AI summary
Provided herein are nucleic acid tags that are linked to, or capable of linking to, a protein of interest. In particular, the nucleic acid tags are oligonucleotides comprising a reporter function and a protein tagging function. Also provided herein, are nucleic acid tag compositions, kits and methods of use thereof.