Single Molecule Sequencing via Chemical Luminescence

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Solution Overview

Problem

Current nucleic acid sequencing technologies face challenges with short read lengths, high error rates, and high costs due to the need for extensive coverage and complex data analysis, as well as polymerase damage from external light excitation and high-intensity luminescence.

Innovation Solution

The method involves a sequencing mixture with a polymerase enzyme, luminescence enzyme, template nucleic acid, and nucleotide-conjugate-analogs with luminescent-substrates, allowing for real-time sequencing through luminescence activation by serial hybridization, where each nucleotide incorporation generates a unique, transient luminescence signal, reducing polymerase damage and requiring less coverage.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If external light excitation and high-intensity luminescence are used for detection, then sequencing signal detection is enabled, but polymerase damage occurs which limits read lengths

Engineering Contradiction:

Improvesequencing signal detectionVSAvoidpolymerase stability

Core Design Contradiction:

Measurement precisionVSReliability

Solution Approach 1:

The patent replaces optical excitation/detection systems with a chemical luminescence system. Instead of using external light to excite fluorescent labels, the invention uses chemically-generated luminescence signals that are detected without external light exposure, thereby eliminating photodamage to the polymerase enzyme while maintaining signal detection capability

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The patent introduces luminescent labels as intermediaries that convert the chemical energy of nucleotide incorporation into detectable light signals. These labels attach to nucleotides and generate luminescence upon incorporation, serving as a bridge between the biochemical reaction and detection without requiring external light exposure that would damage the polymerase

Inventive Principle:

Principle #24Intermediary (Mediator)

2Productivity

If short-read sequencing approaches are used, then sequencing simplicity and speed are improved, but read length is limited and structural variation analysis is not possible

Engineering Contradiction:

Improvesequencing speedVSAvoidread length

Core Design Contradiction:

ProductivityVSLength of stationary object

Solution Approach 1:

The patent employs dynamic, real-time sequencing where the polymerase continuously synthesizes DNA and luminescence signals are detected in real-time without cycle-based interruptions. This dynamic approach allows the polymerase to maintain processivity over long distances, enabling read lengths exceeding 10,000 bases while maintaining sequencing speed through continuous signal generation

Inventive Principle:

Principle #15Dynamics

3Reliability

If high coverage is required to compensate for error rates, then sequencing accuracy is improved, but cost and data analysis complexity increase significantly

Engineering Contradiction:

Improvesequencing accuracyVSAvoiddata analysis complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The patent implements real-time feedback through continuous luminescence monitoring during DNA synthesis. The system detects each nucleotide incorporation event as it occurs, providing immediate feedback on the sequencing process. This real-time detection enables accurate base calling with lower coverage requirements and simplifies data analysis by generating clean, time-resolved signal data that directly corresponds to the sequence being synthesized

Inventive Principle:

Principle #23Feedback

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 longer read lengths with high fidelity and reduced costs by minimizing polymerase damage and the need for extensive coverage, while improving sequencing accuracy and efficiency.

Implementation Method 1

each type of nucleotide-conjugate-analog has a luminescent-substrate attached thereto... the luminescent-substrate is catalyzed by the luminescence-enzyme to produce nucleotide-specific-luminescence

Methodology Applied

Scientific EffectLuminescence: Luminescence

Implementation Method 2

a luminescence enzyme... the luminescent-substrate-attached-leaving-group is combined with the luminescence-enzyme in a luminescence reaction, wherein the luminescence-substrate is catalyzed by the luminescence-enzyme

Methodology Applied

Scientific EffectEnzyme catalysis: Enzyme

Implementation Method 3

carrying out nucleic acid synthesis such that a plurality of nucleotide-conjugate-analogs are added sequentially to the template whereby... the nucleotide-conjugate-analog is incorporated on the template strand by the polymerase

Methodology Applied

Scientific EffectPolymerization:

Data Source

PatentUS20230175054A1Lash methods for single molecule sequencing & target nucleic acid detection

Publication Date: 2023.06.08 SARMAL INC

AI summary

Provided herein are methods and systems for sequencing or detecting a single nucleic acid molecule utilizing components for a luminescence reaction.