Single-Molecule Mutation Detection Using Mismatch Cleavage Probes
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current DNA sequencing methods, including nanopore-based sequencing, are laborious, expensive, and have high error rates, making them impractical for routine diagnostic screening, especially for detecting low-frequency genetic alterations and heterogeneous populations.
Innovation Solution
The method involves using mismatch cleavage probes that form heteroduplexes with target nucleic acids, which are then cleaved at mismatched bases, generating distinct and reproducible signals detectable by nanopores, allowing for single molecule detection and multiplex analysis of mutations or polymorphisms.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If direct DNA sequencing approaches are used, then comprehensive genetic information can be obtained, but the process becomes laborious and expensive
Solution Approach 1:
The invention divides the complex sequencing task into targeted detection of specific mutations or polymorphisms using allele-specific oligonucleotide probes. Instead of sequencing entire genomes, the method segments the analysis to focus only on predetermined genetic variants, dramatically reducing time and cost while maintaining detection accuracy for those specific targets.
Solution Approach 2:
The method performs preliminary enrichment of target nucleic acid sequences before detection through PCR amplification. By pre-amplifying specific genomic regions containing the mutations of interest, the system prepares the sample in advance to enable rapid and accurate detection without requiring comprehensive sequencing of the entire genome.
2Quantity of substance
If next-generation sequencing methods are used, then billions of nucleotides can be sequenced, but error rates increase due to PCR misincorporations
Solution Approach 1:
The invention uses allele-specific oligonucleotide probes that hybridize to complementary sequences, creating a specific binding copy of the target mutation. This hybridization-based copying mechanism is more accurate than PCR amplification because it relies on precise base-pairing recognition rather than enzymatic synthesis, reducing misincorporation errors while still enabling detection of multiple nucleotide variants.
Solution Approach 2:
The method replaces the mechanical/enzymatic PCR amplification process with a chemical hybridization process. Instead of using DNA polymerase to synthesize new strands (which introduces misincorporation errors), the system uses complementary base-pairing chemistry to bind probes to target sequences, providing a more reliable detection mechanism that maintains accuracy even when analyzing large numbers of nucleotides.
3Reliability
If nanopore-based sequencing is used, then amplification steps are eliminated, but translocation speed limits resolution
Solution Approach 1:
The invention introduces nanopore-detectable molecular barcodes as intermediaries that carry mutation information. Instead of directly sequencing DNA through the nanopore (which moves too fast for accurate base calling), the method uses these barcodes as mediators that can be slowly translocated and detected, providing sufficient resolution to distinguish different genetic variants while maintaining the advantage of avoiding PCR amplification.
Solution Approach 2:
The system creates a copy of the genetic information in the form of molecular barcodes that are attached to or associated with the target nucleic acid. These barcodes contain encoded information about the mutation and can be detected by the nanopore at a controlled speed, allowing accurate reading of the genetic variant without requiring fast translocation of the entire DNA molecule.
4Measurement precision
If molecular barcodes are used in nanopore detection, then specific signals can be generated, but separation of bound and unbound fractions becomes critical and complex
Solution Approach 1:
The invention combines the target nucleic acid, the allele-specific probe, and the molecular barcode into a single integrated complex. When the probe hybridizes to its complementary target sequence, the barcode becomes part of the same physical complex, eliminating the need for separate separation steps. The detection system reads the barcode signal directly from this merged complex, simplifying the overall process while maintaining high specificity.
Solution Approach 2:
The system uses the hybridization event itself to generate the detectable signal. When the allele-specific probe binds to its complementary target, the molecular barcode is automatically positioned in a state that generates a detectable nanopore signal. This self-service mechanism means the binding event directly produces the measurement signal without requiring additional separation or processing steps to distinguish bound from unbound molecules.
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 accurate and efficient detection of single molecule nucleic acids, including low-frequency mutations, with reduced error rates and the ability to analyze multiple sequences in a single test sample, overcoming the limitations of existing methods.
Implementation Method 1
The oligonucleotide is capable of hybridizing to the target nucleic acid to form a heteroduplex
Implementation Method 2
contacting the heteroduplexes with a cleavage factor, wherein the cleavage factor is capable of cleaving mismatched bases in the heteroduplexes
Implementation Method 3
The basic concept of nanopore sequencing is to pass a single-stranded DNA molecule through a nanoscale pore embedded in a membrane and measure the ensuing changes in ion current passing through the pore
Data Source
AI summary
Methods and materials are provided for detecting nucleic acid sequence differences including single nucleotide mutations or polymorphisms, one or more nucleotide insertions, and one or more nucleotide deletions in single molecule target members present in a test population of nucleic acid fragments. Heteroduplexes are formed between members of the test nucleic acid population and their corresponding complements provided in a pool of mismatch cleavage probes. Mismatched base pairs in the heteroduplexes are specifically cleaved and cleaved probe fragments are electronically detected to signal the present of the target members in the test population.