Nanopore Detection of Nucleic Acid Sequences
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Solution Overview
Problem
Current diagnostic tests for cancer and infectious diseases face challenges in distinguishing rare or low-abundance mutant sequences from normal cellular markers, leading to false-positive signals and requiring costly, complex sequencing processes that are not cost-effective for early detection or screening.
Innovation Solution
A method involving solid-phase ligase detection reactions, polymerase-mediated extension reactions, and enzymatic cleavage reactions, combined with nanopore-based detection for identifying target nucleotide sequences, transcript sequences, and methylated residues, which eliminates PCR errors, enables digital counting, and reduces costs by eliminating the need for fluorescent labels and thermocycling.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional sequencing processes are used to identify rare mutant sequences, then measurement precision is improved, but device complexity and cost increase significantly
Solution Approach 1:
The detection process is divided into distinct modular steps: capture molecule binding to target nucleic acid, ligase-mediated ligation of identifier sequences, polymerase extension, and nanopore detection. Each step is performed in a separate reaction phase, allowing optimization of individual steps while maintaining overall simplicity and reducing false-positive signals from rare mutant sequences in blood samples
Solution Approach 2:
Capture molecules serve as intermediaries that specifically bind to target nucleic acid sequences from complex biological samples. These capture molecules concentrate rare mutant sequences while excluding abundant normal sequences, enabling sensitive detection without requiring complex sequencing processes. The ligase and polymerase act as additional intermediaries to attach detectable identifiers to the captured targets
2Measurement precision
If PCR amplification is used to enhance signal detection, then measurement precision is improved, but false-positive signals increase due to PCR errors
Solution Approach 1:
The invention extracts and eliminates the PCR amplification step from the detection workflow, replacing it with isothermal ligase-mediated synthesis and nanopore detection. This removal of PCR prevents the introduction of polymerase errors that cause false-positive signals, while still enabling detection of rare mutant sequences through specific capture molecule binding and direct nanopore reading of the amplified or non-amplified target sequences
Solution Approach 2:
The detection method changes the fundamental parameter of nucleic acid amplification from thermal cycling (PCR) to isothermal enzymatic reactions. By maintaining a constant temperature and using ligase-polymerase cascades instead of thermocycling, the method achieves signal amplification without the errors introduced by PCR polymerases, thereby improving reliability while maintaining detection sensitivity
3Measurement precision
If fluorescent labels and thermocycling are used for detection, then measurement precision is improved, but cost and device complexity increase
Solution Approach 1:
The invention replaces the optical detection system (fluorescent labels and microscopy) with an electrical detection system based on nanopore current measurements. This substitution eliminates the need for expensive fluorescent reagents and complex optical instrumentation, reducing both material costs and device complexity while maintaining high detection accuracy through direct electrical reading of nucleic acid sequences as they pass through the nanopore
Solution Approach 2:
The method uses inexpensive, non-fluorescent oligonucleotide identifiers and capture molecules that can be synthesized at low cost. These short-lived reagents are consumed in each reaction but do not require expensive recovery or reuse processes. The nanopore sensor itself is a simple, low-cost component compared to fluorescent detection systems, making the overall assay highly cost-effective for routine clinical screening
4Measurement precision
If multiple markers are examined comprehensively, then measurement precision is improved, but loss of time and cost increase
Solution Approach 1:
The invention merges multiple detection functions into a single integrated nanopore detection platform. Multiple capture molecules targeting different nucleic acid markers can be simultaneously present in the same reaction, and all targets are detected through the same nanopore device using identical isothermal conditions. This consolidation allows comprehensive multi-marker screening in a single assay run, reducing both time and cost compared to performing separate PCR and sequencing reactions for each marker
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 enhances sensitivity and specificity, reduces false-signal detection, and allows for rapid, cost-effective screening and monitoring of cancer and infectious diseases by identifying rare molecular markers in blood samples with high multiplexing power.
Implementation Method 1
The denatured ligation products are fed through one or more nanopores capable of detecting said ligation products
Implementation Method 2
subjecting the immobilized target nucleic acid molecules or immobilized complementary target nucleic acid molecules thereof to a ligase detection reaction to produce ligation products
Implementation Method 3
contacting the immobilized target nucleic acid molecules or immobilized complementary target nucleic acid molecules thereof with a solution to form a nucleotide extension reaction mixture comprising one or more oligonucleotide primers, a polymerase, and a collection of nucleotide triphosphates
Implementation Method 4
contacting the immobilized target nucleic acid molecules or immobilized complementary target nucleic acid molecules thereof with a solution to form a terminal transferase reaction mixture comprising a terminal transferase enzyme and a collection of nucleotide triphosphates
Implementation Method 5
subjecting the immobilized target nucleic acid molecules or immobilized complementary target nucleic acid molecules thereof to a nuclease digestion reaction to produce digested target nucleic acid molecules
Implementation Method 6
identifying, based on said detecting, the presence of one or more target nucleotide sequences differing from other nucleotide sequences in the sample by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and/or one or more methylated residues
Data Source
AI summary
The present invention is directed methods for identifying, in a sample, one or more target nucleotide sequences differing from other nucleotide sequences in the sample by one or more nucleotides, one or more copy numbers, one or more transcript sequences, and/or one or more methylated residues, using ligation detection reactions, polymerase mediated extension reactions, and/or cleavage reactions. The present invention is also directed to methods for identifying, in a sample, one or more nucleotides in a target nucleotide sequence.