Blocking Oligonucleotides for Specific Polynucleotide Detection
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Solution Overview
Problem
Current PCR-based methods for detecting polynucleotide sequences face limitations such as false positives due to non-specific amplification, limited multiplexing capacity, and challenges in quantification accuracy, especially when dealing with low-level analytes and mutations, which affects their specificity and reliability in diagnostic applications.
Innovation Solution
A method involving the use of blocking oligonucleotides in combination with a pyrophosphorolysis and ligation step, where a single-stranded probe oligonucleotide and a pyrophosphorolysing enzyme are used to specifically detect target sequences by creating a partially digested strand that undergoes ligation, enhancing specificity and reducing non-specific binding.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If PCR-based methods are used for detecting polynucleotide sequences, then amplification sensitivity is improved, but false positives occur due to non-specific amplification
Solution Approach 1:
The patent introduces blocking oligonucleotides as intermediary elements that specifically bind to non-target sequences, preventing them from participating in non-specific amplification. These blocking oligos act as mediators between the primer and the non-target DNA, allowing the primer to distinguish between target and non-target sequences by blocking the latter while leaving the former accessible for specific amplification.
2Adaptability or versatility
If multiplexing of PCR-based methods is increased, then detection capacity is improved, but primer-primer interactions increase resulting in limited operational windows
Solution Approach 1:
The patent applies preliminary anti-action by adding blocking oligonucleotides before the PCR amplification step. These blocking oligos pre-bind to potential non-target sequences and prevent primer-primer interactions that would otherwise occur during multiplexing. By addressing potential problems before they arise, the method enables higher multiplexing capacity without the usual increase in primer interaction complexity.
3Productivity
If PCR reaction cycles exponentially, then amplification efficiency is improved, but quantification accuracy deteriorates due to small variations in reaction efficiency
Solution Approach 1:
The blocking oligonucleotides serve as intermediaries that stabilize the amplification process by preventing non-specific binding events that would otherwise cause variations in reaction efficiency. By ensuring that only target sequences are amplified through specific primer binding, the blocking oligos reduce the impact of small variations in reaction conditions, thereby improving quantification accuracy while maintaining exponential amplification efficiency.
4Adaptability or versatility
If mutations occur in the targeted region, then detection robustness is improved against some variants, but false negatives or false positives increase depending on the mutation type
Solution Approach 1:
The patent employs parameter changes by designing blocking oligonucleotides with specific sequences, concentrations, and binding affinities that can be adjusted to account for different mutation types. By varying these parameters, the method can adapt to detect or block specific mutant variants while maintaining accuracy. The blocking oligos can be designed to tolerate certain mutations in the target sequence while still effectively blocking non-target sequences, thus improving both mutation tolerance and detection accuracy.
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 improves the specificity and accuracy of polynucleotide sequence detection, reducing false positives and enabling the reliable identification of target sequences even at low concentrations, thereby enhancing the reliability of diagnostic tests.
Implementation Method 1
a pyrophosphorolysing enzyme; wherein the target analyte anneals to the single-stranded probe oligonucleotide A0 to create a first intermediate product which is at least partially double-stranded and in which the 3′ end of A0 forms a double-stranded complex and A0 is pyrophosphorolysed in the 3′-5′ direction from the 3′ end
Implementation Method 2
a ligase; wherein the target analyte anneals to the single-stranded probe oligonucleotide A0 to create a first intermediate product which is at least partially double-stranded and in which the 3′ end of A0 forms a double-stranded complex and A0 is pyrophosphorolysed in the 3′-5′ direction from the 3′ end to create at least a partially digested strand A1 and A1 undergoes ligation to form A2
Implementation Method 3
introducing a blocking oligonucleotide to a first reaction mixture comprising one or more nucleic acid analytes, wherein the blocking oligonucleotide anneals to at least a subset of non-target polynucleotide sequences
Data Source
AI summary
This invention relates to simplified polynucleotide sequence detection methods suitable for testing for the presence of a large number of diagnostic markers, including those used in the identification of cancer, infectious disease and transplant organ rejection. It is also useful for companion diagnostic testing in which a panel of markers must be identified reliably and at low cost.