Nanopore Sequencing via Tag Release for Base Differentiation

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Solution Overview

Problem

Current nucleic acid sequencing methods using single-stranded molecules passing through nanopores lack sensitivity due to insufficiently distinct signals between nucleic acid bases, particularly between purines (A and G) and pyrimidines (C, T, U), which hinders accurate identification and sequencing.

Innovation Solution

A method involving a chip with individually addressable nanopores linked to electrodes and nucleic acid polymerases, where tagged nucleotides are polymerized to release tags that pass through the nanopores, allowing for accurate detection and sequencing by distinguishing between individual nucleotide incorporation events with high accuracy.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If single-stranded nucleic acid molecules are used to pass through nanopores for sequencing, then the sequencing process can be performed, but the sensitivity is insufficient due to indistinct signals between nucleic acid bases

Engineering Contradiction:

Improvesignal distinction between nucleic acid basesVSAvoidsequencing sensitivity

Core Design Contradiction:

Measurement precisionVSReliability

Solution Approach 1:

The patent introduces an intermediary detection mechanism where tagged nucleotides are polymerized and the released tags are detected by nanopores. The tag acts as a mediator that translates the nucleotide incorporation event into a detectable signal, resolving the indistinctness problem of direct nucleic acid base detection through nanopores.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent changes the detection parameter from directly measuring nucleic acid bases to measuring the tags released during polymerization. This parameter transformation enables distinct signal detection for different nucleotide types, improving measurement precision and sequencing reliability.

Inventive Principle:

Principle #35Parameter changes

2Measurement precision

If tagged nucleotides are polymerized with nanopore detection, then sequencing accuracy is improved, but the device complexity increases

Engineering Contradiction:

Improvenucleotide identification accuracyVSAvoidchip and nanopore system complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The patent segments the sequencing function into distinct components: polymerization occurs at one location while nanopore detection occurs at another. This segmentation allows each component to be optimized independently, managing device complexity while maintaining high measurement precision through specialized detection of released tags.

Inventive Principle:

Principle #1Segmentation

3Measurement precision

If tags are released from tagged nucleotides during polymerization, then nucleotide differentiation is enabled, but the process time increases

Engineering Contradiction:

Improvenucleotide base differentiationVSAvoidpolymerization and detection process time

Core Design Contradiction:

Measurement precisionVSLoss of time

Solution Approach 1:

The patent maintains continuous useful action by having the tag release and detection occur in an integrated manner during polymerization. The nanopore detection of released tags happens concurrently with the polymerization process, enabling nucleotide differentiation without significant time loss while maintaining measurement precision.

Inventive Principle:

Principle #20Continuity of useful action

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 the sensitivity and accuracy of nucleic acid sequencing by enabling the differentiation of nucleotide bases with an accuracy greater than 4σ, overcoming the limitations of existing methods by using tagged nucleotides and nanopore detection to identify and sequence nucleic acids effectively.

Implementation Method 1

detecting the tag with the aid of said electrode, wherein the tag is detected subsequent to being released from said individual nucleotide

Methodology Applied

Scientific EffectIonic current measurement: Conduction (electrical)

Implementation Method 2

with the aid of said polymerase, polymerizing nucleotides along said nucleic acid molecule to generate a strand that is complementary to at least a portion of said nucleic acid molecule

Methodology Applied

Scientific EffectEnzyme catalysis: Enzyme

Data Source

PatentUS11795191B2Method of preparation of nanopore and uses thereof

Publication Date: 2023.10.24 THE TRUSTEES OF COLUMBIA UNIV IN THE CITY OF NEW YORK

AI summary

This disclosure provides systems and methods for sequencing nucleic acids using nucleotide analogues and translocation of tags from incorporated nucleotide analogues through a nanopore. In aspects, this disclosure is related to composition, method, and system for sequencing a nucleic acid using tag molecules and detection of translocation through a nanopore of tags released from incorporation of the molecule.