Nucleic Acid Variant Detection Using Selector Blockers
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Solution Overview
Problem
Current methods for detecting rare nucleic acid sequence variants suffer from poor enzyme fidelity, leading to errors that cannot be distinguished from true variants, limiting sensitivity and specificity, especially in amplification methods using DNA polymerases which introduce errors, resulting in an intrinsic background noise.
Innovation Solution
The use of a selector blocker in conjunction with primers to amplify a target region, where the selector blocker prevents extension in the presence of wild-type sequences, allowing for the detection of nucleic acid variants such as deletions, mutations, or insertions with high sensitivity by differentiating amplification signals between the presence and absence of variants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If DNA polymerase amplification is used to detect rare nucleic acid variants, then amplification sensitivity is improved, but enzyme fidelity deteriorates leading to errors that cannot be distinguished from true variants
Solution Approach 1:
The amplification process is divided into two distinct stages: a first amplification reaction that generates sufficient product, and a second amplification reaction that provides high-fidelity copying. This segmentation allows each stage to be optimized for its specific purpose, resolving the contradiction between sensitivity and fidelity.
Solution Approach 2:
The first amplification reaction performs preliminary copying to generate enough target product from rare variants, while the second high-fidelity reaction then copies this product. The preliminary action of the first reaction enables the subsequent high-fidelity reaction to work on already-amplified material, achieving both sensitivity and accuracy.
2Measurement precision
If allele-specific priming methods are used to detect variants, then specificity is improved, but mispriming occurs during amplification that overwrites variant sites
Solution Approach 1:
The first amplification reaction performs preliminary copying using allele-specific priming to enrich variant-containing products. The second high-fidelity reaction then performs accurate copying of these enriched products, preventing mispriming errors from propagating and overwriting variant sites.
Solution Approach 2:
The first amplification reaction acts as an intermediary that enriches variant-containing products before they enter the second high-fidelity reaction. This intermediary step separates the specificity function from the fidelity function, allowing each to be optimized independently.
3Productivity
If standard PCR amplification is used to amplify target regions, then amplification efficiency is improved, but polymerase errors are introduced that create background noise
Solution Approach 1:
The amplification process is segmented into two reactions: the first reaction prioritizes efficiency and productivity to generate sufficient product, while the second reaction prioritizes reliability and sequence accuracy. This segmentation resolves the contradiction by assigning different optimization goals to different stages.
Solution Approach 2:
The first efficient amplification reaction performs preliminary product generation, creating enough material for the second high-fidelity reaction to work with. This preliminary action allows the second reaction to focus on accuracy without being constrained by efficiency requirements.
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 enables the detection of 1 copy of a nucleic acid variant in the presence of 1000 copies of wild-type sequences, achieving a sensitivity of >1:1000 and higher, with minimal interference from wild-type amplification, thereby improving the specificity and accuracy of variant detection.
Implementation Method 1
The selector blocker includes a sequence complementary to the target region in the absence of the nucleic acid variant
Implementation Method 2
the bound selector blocker portion of the primer-switch functions as a steric blocker preventing extension of the forward primer
Implementation Method 3
polymerization by DNA polymerase are coupled serially by utilizing a pyrophosphorolysis-activatable oligonucleotide (P*)
Implementation Method 4
amplifying the target region with a forward primer and a reverse primer in the presence of a selector blocker
Data Source
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AI summary
The present invention provides methods for detecting the presence or absence of a nucleic acid variant in a target region. These methods include amplifying the target region with a forward primer and a reverse primer in the presence of a selector blocker. The selector blocker includes a sequence complementary to the target region in the absence of the nucleic acid variant. The methods further include detecting amplification of the target region where amplification of the target region indicates the presence of the nucleic acid variant in the target region. The nucleic acid variant can include deletions, mutations or insertions.