Non-Extending Helper Oligonucleotide for Nucleic Acid Amplification
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Solution Overview
Problem
Current nucleic acid amplification and detection methods, such as PCR, face challenges in achieving efficient and sensitive detection of target nucleic acids, particularly for low copy numbers, due to limitations in primer binding and secondary structure stability.
Innovation Solution
The use of a non-extending helper oligonucleotide that anneals to the target nucleic acid but does not extend, reducing the Gibbs Free Energy of the secondary structure and enhancing primer accessibility, thereby improving amplification efficiency and sensitivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If standard PCR amplification is used, then amplification can be performed, but amplification efficiency is insufficient for detecting low copy numbers of target nucleic acid
Solution Approach 1:
The patent introduces a non-extending helper oligonucleotide as an intermediary component that binds to the target nucleic acid and facilitates primer binding. This helper oligonucleotide acts as a mediator that enhances the interaction between the primer and target, thereby improving amplification efficiency and detection sensitivity for low copy number targets without interfering with the standard PCR process
Solution Approach 2:
The patent modifies the Gibbs Free Energy of the secondary structure by introducing the non-extending helper oligonucleotide, which changes the thermodynamic parameters of the reaction system. This parameter change optimizes the binding affinity and stability of the primer-target complex, enabling more efficient amplification of low copy number targets
2Productivity
If primers are used for amplification, then nucleic acid can be amplified, but primer accessibility to target nucleic acid is limited by secondary structure stability
Solution Approach 1:
The non-extending helper oligonucleotide serves as an intermediary that binds to the target nucleic acid in regions that may be obscured by secondary structures. By occupying these regions and stabilizing the target conformation, the helper oligonucleotide makes the primer binding sites more accessible, thereby improving amplification efficiency without requiring changes to the primer design or reaction conditions
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 leads to increased accumulation of PCR products and improved sensitivity, enabling the detection of small amounts of target nucleic acid, even in complex samples, by facilitating primer binding and extension.
Implementation Method 1
reducing the Gibbs Free Energy of the secondary structure and enhancing primer accessibility
Implementation Method 2
a non-extending helper oligonucleotide that anneals to the target nucleic acid but does not extend
Implementation Method 3
PCR utilizes a polymerase enzyme
Data Source
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AI summary
Improved methods for the detection and quantitation of a target nucleic acid in a sample using a non-extending helper oligonucleotide are described. The methods include contacting nucleic acids in a sample with amplification reagents including one or more primers, one or more non-extending helper oligonucleotides, and one or more probes. The non-extending helper oligonucleotide facilitates and increases the target nucleic acid accessibility of one or more of the primers, result in greater accumulation of amplicon production, thereby increasing the efficiency and sensitivity of the amplification assay, including amplification assays for Hepatitis C Virus (HCV), for example, HCV Genotype 5. Kits, articles of manufacture, and reaction mixtures are also provided.