BNA Clamp Nucleic Acid for Trace Mutant Gene Detection
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Solution Overview
Problem
Current methods for detecting mutated genes in somatic cells, especially when they are present in trace amounts compared to wild-type genes, suffer from low sensitivity and accuracy, making it difficult to differentiate between the two effectively.
Innovation Solution
The use of second-generation BNAs as clamp nucleic acids in nucleic acid amplification techniques, combined with a detection probe, allows for the selective amplification and detection of mutated genes even when they are present in very small amounts, enhancing detection sensitivity to 0.01% or lower.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional detection methods (dideoxysequencing, pyrosequencing, melting curve analysis) are used, then detection can be performed, but detection sensitivity is limited to about 5% or 10% for mutated genes in wild-type genes
Solution Approach 1:
The clamp nucleic acid selectively binds to and extracts the wild-type gene sequences, removing them from the amplification reaction. This allows the mutated genes, which are not bound by the clamp, to be selectively amplified and detected, thereby extracting the signal of interest from the background of abundant wild-type sequences
Solution Approach 2:
The clamp nucleic acid acts as an intermediary molecule that mediates the selective inhibition of wild-type gene amplification. By designing the clamp to be complementary to the wild-type sequence but mismatched with the mutated sequence, it serves as a selective mediator that blocks amplification of the dominant wild-type background while allowing mutated variants to be detected
2Measurement precision
If mutant-enriched PCR with restriction enzyme digestion is used, then detection sensitivity can reach 1 molecule in 10^6 wild-type genes, but the operation becomes highly complicated
Solution Approach 1:
The clamp nucleic acid directly extracts and inhibits wild-type gene amplification through selective hybridization, eliminating the need for restriction enzyme digestion and multiple processing steps. This simplifies the workflow while maintaining high detection sensitivity
Solution Approach 2:
The method changes the fundamental parameter of amplification selectivity by using clamp-mediated inhibition rather than enzymatic digestion. This parameter change transforms a multi-step complex procedure into a simple one-step PCR reaction with added clamp nucleic acid
3Measurement precision
If ARMS or ASPCR methods are used, then detection sensitivity is comparatively high and operations are convenient, but false-positivity risk increases when wild-type gene is amplified due to erroneous single base distinction
Solution Approach 1:
The method converts the harmful effect of abundant wild-type genes (which cause false-positives in ARMS/ASPCR) into a beneficial feature. By using the clamp to selectively inhibit wild-type amplification, the dominant background sequence becomes the target of inhibition rather than amplification, thereby eliminating false-positives while maintaining sensitivity
Solution Approach 2:
The clamp nucleic acid serves as an intermediary that mediates selective inhibition between the primer and the wild-type template. This intermediary layer provides an additional level of specificity control, reducing false-positives by preventing wild-type amplification before the primer can bind erroneously
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 highly sensitive and accurate detection of mutated genes, including point mutations, deletions, and insertions, in a cost-effective manner, even in samples predominantly containing wild-type genes.
Implementation Method 1
a step of selectively amplifying a region containing at least a part of the detection target site of the detection target nucleic acid in a test sample by a nucleic acid amplification method using a clamp nucleic acid having a base sequence complementary to the base sequence of the detection target site in the detection non-target nucleic acid
Data Source
AI summary
The invention provides a method of selectively amplifying a detection target nucleic acid by inhibiting amplification of a detection non-target nucleic acid (e.g., wild-type gene) in a test sample by using, in a nucleic acid amplification reaction, an oligonucleotide analog containing one or more kinds of one or more unit structures of various nucleoside analogs represented by the following formula (I):wherein the symbols are as defined in the DESCRIPTION, and the like, or a salt thereof, as a clamp nucleic acid, and detecting the amplified nucleic acid.