Oligonucleotide Tag Sequence Selection for Nucleic Acid Assays
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Solution Overview
Problem
Current nucleic acid assays face challenges with the lack of a rigorous method for selecting optimal tag sequences, leading to suboptimal performance due to undesired interactions between tags, primers, and other nucleic acid sequences, which results in reduced assay accuracy and precision, especially in multiplex amplification reactions.
Innovation Solution
A method for identifying and selecting unique nucleic acid tag sequences that minimize interference by generating a pool of sequences, screening for specific performance characteristics, and synthesizing oligonucleotides with optimized tag sequences to reduce cross-reactions and enhance assay performance.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If user-selected tag sequences are used in nucleic acid assays, then assay flexibility and customization are improved, but undesired nucleic acid interactions increase leading to reduced assay accuracy and precision
Solution Approach 1:
The patent applies preliminary action by performing in silico screening of tag sequences before actual assay execution. The method screens candidate tags against databases of assay sequences to predict and prevent undesired interactions in advance, selecting optimized tags that minimize cross-reactions while maintaining assay flexibility.
Solution Approach 2:
The patent introduces an intermediary computational screening process between tag selection and assay execution. This in silico screening acts as a mediator that evaluates potential tag sequences against assay-specific sequence databases, filtering out tags that would cause harmful interactions before they can affect assay performance.
2Productivity
If multiple tag sequences are used in multiplex amplification reactions, then detection capability for multiple targets is improved, but cross-reactions between tags and other nucleic acid sequences increase
Solution Approach 1:
The patent performs preliminary in silico screening of multiple candidate tag sequences against comprehensive databases containing all assay sequences including primers, probes, and target sequences. This advance evaluation identifies tags that minimize cross-reactions while maintaining multiplex detection capability.
Solution Approach 2:
The computational screening process serves as an intermediary that evaluates each candidate tag's potential for cross-reactions with other nucleic acid sequences in the multiplex assay. This mediator filters out problematic tags before assay execution, preventing cross-reactions from occurring.
3Loss of time
If standard tag sequences are used without optimization, then assay development time is reduced, but assay performance deteriorates due to undesired interactions
Solution Approach 1:
The patent performs preliminary in silico optimization of tag sequences before assay execution. This advance computational screening and selection process identifies optimal tags that minimize undesired interactions, ensuring high assay performance while maintaining efficient development timelines.
Solution Approach 2:
The patent changes the selection criteria for tag sequences from arbitrary or standard choices to optimized selections based on in silico screening results. By evaluating tags against assay-specific sequence databases and selecting tags with optimal characteristics, the method improves assay reliability without significantly extending development time.
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
The method effectively reduces interference between nucleic acid sequences, improving the accuracy and precision of nucleic acid assays by using uniquely designed tag sequences that do not interact with each other or other sequences, thereby enhancing the dynamic range and sensitivity of target nucleic acid detection.
Implementation Method 1
screening said pool of nucleic acid sequences to identify two or more nucleic acid sequences having two or more performance characteristics including minimal complementarity to nucleic acid sequences in said assay
Data Source
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AI summary
There is disclosed a method for identifying nucleic acid tag sequences for use in an in vitro nucleic acid amplification assay, comprising the steps of: a) generating a pool of nucleic acid sequences, wherein the pool is at least three nucleic acid sequences from Table 1; b) screening the pool of nucleic acid sequences against a database containing one or more nucleic acid sequences to identify percent complementarity between nucleic acid sequences in the pool and nucleic acid sequences in the database; c) screening the pool of nucleic acid sequences to determine a performance characteristic selected from the group consisting of: G-C content, nucleobase composition, length, multimer formation, primer-dimer formation, Tm, hairpin stabilization energy, self-dimer stabilization energy, internal structure formation, G-quartet formation, hybridization energy, activity in an enzyme reaction, and combinations thereof; d) generating a sub-pool of nucleic acid sequences from the results obtained in step b), step c) or steps b) and c); and e) selecting one or more nucleic acid sequences from the sub-pool for use as tag sequences in a nucleic acid assay.