Blocker Oligonucleotides for Selective Nucleic Acid Amplification
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Solution Overview
Problem
Existing nucleic acid amplification systems face challenges with false positive results due to contaminating nucleic acid and require stringent purification and sterility efforts, limiting their acceptance in clinical and other settings.
Innovation Solution
The development of methods and reaction mixtures that use tagged oligonucleotides and blockers to selectively amplify target nucleic acid sequences, reducing false positives by stabilizing oligonucleotides and preventing their hybridization to the target sequence, allowing for less stringent purification and sterility requirements.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional nucleic acid amplification systems are used, then amplification can be performed, but false positive results occur due to contaminating nucleic acid requiring stringent purification and sterility efforts
Solution Approach 1:
The patent introduces a blocker oligonucleotide as an intermediary substance that binds to contaminating nucleic acid sequences, preventing them from acting as templates in the amplification reaction. This mediator selectively interferes with harmful contaminants while allowing legitimate target sequences to be amplified, thereby reducing false positives without requiring extensive purification steps
Solution Approach 2:
The blocker oligonucleotide is added to the reaction mixture before amplification occurs, performing preliminary neutralization of potential contaminants. By pre-binding to contaminating nucleic acid sequences, the blocker prevents them from causing false positive amplification signals, thus proactively protecting the assay accuracy before the harmful effect can manifest
2Reliability
If stringent purification and sterility efforts are implemented, then false positive results are reduced, but the complexity and time required for sample preparation increase
Solution Approach 1:
The patent extracts the function of contamination prevention from the sample preparation stage and relocates it to the amplification reaction stage. Instead of spending time and effort purifying samples beforehand, the blocker oligonucleotide is introduced during amplification to selectively neutralize contaminants, thereby reducing both preparation time and complexity while maintaining reliability
3Reliability
If blocker oligonucleotides are used to prevent contaminating sequence amplification, then false positive signals are reduced, but the reaction mixture complexity increases
Solution Approach 1:
The blocker oligonucleotide is designed as a simple, inexpensive molecular component that performs its function temporarily during the amplification reaction and then becomes irrelevant. Unlike complex purification systems, the blocker is a single-use reagent that adds minimal complexity to the reaction mixture while effectively preventing false positives through its temporary binding action on contaminants
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
These methods effectively reduce false positive amplification signals and minimize the need for stringent purification and sterility efforts, enhancing the accuracy and reliability of nucleic acid detection in various samples, including clinical and environmental samples.
Implementation Method 1
providing a blocker oligonucleotide to the sample under conditions permitting the blocker oligonucleotide to stably hybridize to a region of a nucleic acid containing the target nucleic acid sequence
Data Source
AI summary
A method for the selective amplification of a target sequence. The method includes hybridizing a tagged oligonucleotide to the target sequence and reducing the effective concentration of unhybridized tagged oligonucleotide which is capable of hybridizing to the target sequence. The tagged oligonucleotide includes a target hybridizing sequence and a tag sequence situated 5′ to target hybridizing sequence, where the tag sequence does not stably hybridize to a target nucleic acid containing the target sequence. A blocker oligonucleotide is provided which is designed to hybridize to a region of a nucleic acid containing the target sequence, where the region targeted by the blocker oligonucleotide is 3′ to the target sequence. Amplification products are formed using first and second oligonucleotides. The first oligonucleotide hybridizes to the 3′-end of the complement of the target sequence, and the second oligonucleotide hybridizes to the complement of the tag sequence.