Pyrrole-Imidazole Polyamide Nucleic Acid Concentration
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Solution Overview
Problem
Current methods for concentrating target double-stranded nucleic acid molecules, especially those with one or more base differences, face challenges in discrimination and amplification, leading to false-positive results and require strict temperature control, which limits their effectiveness in genetic diagnosis, particularly in samples with low amounts of mutated genes like cancer cells.
Innovation Solution
A method using pyrrole-imidazole-containing polyamides modified with linker molecules and ligands to specifically bind and separate target double-stranded nucleic acid molecules from non-target molecules, allowing for their concentration and removal, thereby enhancing the ratio of mutated genes in a sample for accurate genetic diagnosis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If PCR clamp method is used to concentrate mutated genes, then the ratio of mutated genes is increased, but false-positive results occur due to base insertion by Taq polymerase non-fidelity
Solution Approach 1:
The patent introduces a pyrrole-imidazole-containing polyamide as an intermediary substance that specifically binds to mutated double-stranded nucleic acid sequences. This polyamide acts as a mediator between the mutated genes and the separation system, enabling selective concentration without relying on PCR amplification that causes base insertion errors. The polyamide binds to the minor groove of target DNA sequences with high specificity, allowing accurate enrichment of mutated genes while maintaining diagnosis reliability.
2Quantity of substance
If PCR amplification is used to increase the amount of target genes, then the quantity of genes is increased, but the ratio of mutated genes in the sample is not improved
Solution Approach 1:
The patent extracts and separates mutated double-stranded nucleic acid molecules from the complex biological sample using pyrrole-imidazole-containing polyamides. By specifically binding to mutated sequences and separating them from normal cells and other components, the method both increases the quantity of detectable target genes and improves the ratio of mutated genes in the final concentrate, solving the limitation of PCR amplification which only increases total amount without improving purity.
3Manufacturing precision
If PCR clamp method is used for gene concentration, then sequence specificity is achieved, but strict temperature control and complex reaction system control are required
Solution Approach 1:
The patent replaces the complex thermal control system required by PCR clamp method with a chemical binding system based on pyrrole-imidazole-containing polyamides. Instead of relying on temperature cycling and precise thermal control to achieve sequence specificity, the method uses the inherent chemical affinity and specificity of polyamides for binding to mutated DNA sequences at constant temperature, thereby maintaining high sequence specificity while eliminating the need for complex temperature control apparatus.
4Quantity of substance
If normal cells are included in cancer samples, then the sample amount is sufficient, but the correctness of genetic mutation detection decreases
Solution Approach 1:
The patent converts the presence of normal cells, which is normally a harmful factor diluting the mutated genes, into a beneficial situation. By using pyrrole-imidazole-containing polyamides that specifically recognize and bind only to mutated sequences, the method selectively extracts mutated genes even from samples dominated by normal cells. The normal cells serve as a background matrix from which the mutated genes are specifically isolated, thereby improving mutation detection correctness while maintaining sufficient sample amount.
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 method enables efficient concentration and separation of target nucleic acid molecules, reducing false positives and eliminating the need for strict temperature control, improving the accuracy of genetic diagnosis even in samples with low amounts of mutated genes.
Implementation Method 1
a pyrrole-imidazole-containing polyamide (first PI polyamide) modified with a first linker molecule and capable of specifically binding to a sequence of the target double-stranded nucleic acid molecule
Implementation Method 2
a carrier a modified with a first ligand molecule capable of specifically binding and/or adsorbing to the first linker molecule
Data Source
AI summary
A method of separating a target double-stranded nucleic acid molecule from a sample including the target double-stranded nucleic acid molecule and a non-target double-stranded nucleic acid molecule, including (1) mixing the sample, a pyrrole-imidazole-containing polyamide (first PI polyamide) modified with a first linker molecule and capable of specifically binding to a sequence of the target double-stranded nucleic acid molecule, and a carrier a modified with a first ligand capable of specifically binding and/or adsorbing to the first linker molecule such that a mixed solution is produced, (2) forming a complex A by binding the carrier a to the first PI polyamide with which the target double-stranded nucleic acid molecule is bound in the mixed solution, and (3) separating the complex A from the mixed solution.