Modified Nucleotides for Nanopore DNA Sequencing

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Solution Overview

Problem

Current DNA sequencing technologies, particularly those using nanopores, have not achieved base-to-base sequencing of DNA, struggling to distinguish between individual nucleotides such as A, C, G, and T due to similar electronic signatures generated by these bases when passing through a nanopore.

Innovation Solution

Modifying nucleotides by attaching specific groups such as azido or amino moieties to differentiate their electronic signatures, allowing for distinguishable signals when passing through a nanopore, enabling the determination of nucleotide sequences by comparing these signatures.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If standard nucleotides are used in nanopore sequencing, then the method is simple and direct, but the electronic signatures of different nucleotides are similar and cannot be reliably distinguished

Engineering Contradiction:

Improvenucleotide identification accuracyVSAvoidnucleotide modification complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The patent applies local quality modification by attaching specific modifying groups to specific nucleotides (A, C, G, or T) at particular positions in the DNA sequence. This creates locally distinct electronic signatures for different nucleotides while maintaining the overall simplicity of the nanopore sequencing method. The modifying groups are strategically placed to differentiate nucleotide types without requiring complex modifications throughout the entire DNA molecule.

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The patent changes the physical-chemical parameters of the nucleotides by introducing modifying groups with different sizes, shapes, and charge distributions. These parameter changes create distinct electronic signatures that can be detected by the nanopore, enabling reliable differentiation between nucleotide types based on their modified physical properties rather than inherent similarities.

Inventive Principle:

Principle #35Parameter changes

2Measurement precision

If modifying groups are attached to nucleotides to create distinguishable electronic signatures, then nucleotide identification accuracy improves, but the complexity of nucleotide preparation increases

Engineering Contradiction:

Improvebase-to-base sequencing accuracyVSAvoidmodified nucleotide synthesis complexity

Core Design Contradiction:

Measurement precisionVSEase of manufacture

Solution Approach 1:

The patent employs preliminary action by pre-attaching modifying groups to nucleotides before they are incorporated into the DNA sequence. This allows the modifying groups to be in place before sequencing, eliminating the need for complex in-situ modification during the sequencing process. The modifying groups are introduced in advance through standardized chemical synthesis methods, simplifying the overall manufacturing process.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The patent uses modifying groups as intermediary elements that mediate between the nucleotide base and the detection system. These modifying groups serve as recognizable markers that translate the inherent similarities of standard nucleotides into distinguishable electronic signatures, enabling accurate sequencing without requiring direct detection of the base pairs themselves.

Inventive Principle:

Principle #24Intermediary (Mediator)

3Measurement precision

If standard nucleotides are used, then the sequencing process is straightforward, but the ability to distinguish between similar nucleotides like A and G is insufficient

Engineering Contradiction:

Improvenucleotide differentiation capabilityVSAvoidsignal processing complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The patent addresses the difficulty of distinguishing similar nucleotides by applying local quality modifications specifically to nucleotides that are difficult to differentiate. By attaching distinct modifying groups to specific nucleotide positions, the method creates locally unique electronic signatures that simplify the differentiation process rather than requiring complex global signal analysis.

Inventive Principle:

Principle #3Local quality

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

Enables accurate determination of nucleotide sequences by enhancing the size and charge differences between nucleotides, allowing for reliable base-to-base sequencing through distinct electronic signatures generated during passage through a nanopore.

Implementation Method 1

The limiting aperture of the nanopore allows linear single-stranded but not double-stranded nucleic acid molecules (diameter −2.0 nm) to pass through. The polyanionic nucleic acids are driven through the pore by the applied electric field, which blocks or reduces the ionic current that would be otherwise unimpeded.

Methodology Applied

Scientific EffectIonic current blockade: Electrical Resistance