FRET-Based DNA Sequencing Resolving Throughput Accuracy Trade-Off

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Solution Overview

Problem

Current high-throughput DNA sequencing technologies face challenges such as high costs, biased coverage of GC-rich or AT-rich genomes, inability to accurately sequence homopolymer stretches, difficulty in sequencing beyond 200 nucleotides, and limited throughput, which hinder the achievement of low-cost, rapid, and highly sensitive sequencing.

Innovation Solution

A method utilizing Förster Resonance Energy Transfer (FRET) between fluorescence donor and acceptor molecules attached to nucleotide analogues and DNA polymerase, allowing for the determination of nucleotide identity and sequence through the formation of ternary complexes and iterative incorporation of nucleotide analogues, enabling accurate and efficient sequencing.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If current next-generation sequencing platforms are used, then throughput is improved, but cost remains prohibitive and accuracy is insufficient

Engineering Contradiction:

Improvesequencing throughputVSAvoidsequencing accuracy

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent replaces traditional mechanical/optical detection methods with FRET-based energy transfer detection. By using fluorophore-labeled nucleotides that transfer energy to a detector upon incorporation, the system achieves higher accuracy in detecting nucleotide identity while maintaining high throughput, directly resolving the contradiction between throughput and accuracy

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The patent changes the detection parameter from direct fluorescence emission to FRET energy transfer efficiency. By monitoring the efficiency of energy transfer between donor and acceptor fluorophores, the system can distinguish nucleotide incorporations with higher precision, thereby improving sequencing accuracy without sacrificing throughput

Inventive Principle:

Principle #35Parameter changes

2Length of moving object

If sequencing length is extended beyond 200 nucleotides, then more complete genome coverage is achieved, but sequencing accuracy deteriorates

Engineering Contradiction:

Improvesequenced DNA lengthVSAvoidnucleotide identification accuracy

Core Design Contradiction:

Length of moving objectVSMeasurement precision

Solution Approach 1:

The patent introduces FRET as an intermediary detection mechanism that allows indirect observation of nucleotide incorporation. The energy transfer signal provides a amplified and more reliable detection method that maintains accuracy over longer sequencing distances, enabling extension beyond 200 nucleotides while preserving measurement precision

Inventive Principle:

Principle #24Intermediary (Mediator)

3Measurement precision

If FRET-based detection with fluorophore-labeled nucleotides is used, then sequencing accuracy is improved, but reagent cost increases

Engineering Contradiction:

Improvenucleotide incorporation detection accuracyVSAvoidcost of fluorophore-labeled nucleotide reagents

Core Design Contradiction:

Measurement precisionVSQuantity of substance

Solution Approach 1:

The patent designs fluorophore-labeled nucleotides that serve multiple functions: they act as both the nucleotide substrate for DNA synthesis and the detection probe for sequencing. This dual functionality eliminates the need for separate labeling steps and reduces overall reagent consumption, thereby lowering costs while maintaining high detection accuracy

Inventive Principle:

Principle #6Universality (Multi-functionality)

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 sequencing accuracy and throughput while reducing costs, enabling the sequencing of longer DNA strands and improving coverage of challenging genomic regions, moving closer to the goal of the '$1,000 Genome' by leveraging FRET-based nucleotide detection.

Implementation Method 1

The underlying photophysical principle for this SBS method is based on Förster resonance energy transfer (FRET), where the energy of electronic excited states of a donor molecule is transferred to an acceptor molecule via non-radiative dipole-dipole interactions.

Methodology Applied

Scientific EffectFörster Resonance Energy Transfer (FRET):

Implementation Method 2

the luminescence of the donor molecule is quenched and fluorescence of the acceptor molecule is observed

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Data Source

PatentUS11384391B2Flourescene energy transfer-based single molecule/ensemble DNA sequencing by synthesis

Publication Date: 2022.07.12 THE TRUSTEES OF COLUMBIA UNIV IN THE CITY OF NEW YORK

AI summary

This invention provides nucleotide analogues each of which comprises a tag comprising one or more Forster resonance energy transfer (FRET) acceptor fluorophores, a nucleotide polymerase having one or more FRET donor fluorophores, and methods for sequencing single-stranded.