FRET DNA Sequencing by Synthesis for Long, Accurate Reads
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current DNA sequencing technologies face challenges with high costs, biased coverage of GC-rich or AT-rich regions, inability to sequence through homopolymers, difficulty in sequencing beyond 200 nucleotides, and limited throughput, which hinder applications in personalized medicine and genomic studies.
Innovation Solution
A method utilizing Förster Resonance Energy Transfer (FRET) for sequencing by synthesis (SBS) by forming a ternary complex with DNA polymerase, donor fluorophores, and acceptor dyes on nucleotide analogues, enabling accurate and efficient nucleotide identification and sequencing through fluorescence emissions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current next-generation sequencing platforms are used, then sequencing cost is reduced below $10,000 per genome, but the cost remains prohibitive for most laboratories and clinical applications
Solution Approach 1:
The patent employs disposable microfluidic cartridges containing all necessary reagents and components for a single sequencing run. This eliminates the need for expensive reusable instrumentation and complex reagent preparation systems, making sequencing accessible to smaller laboratories and clinical settings while maintaining automated performance.
Solution Approach 2:
The system utilizes self-contained microfluidic cartridges that automatically perform all sequencing operations including sample preparation, nucleotide incorporation, and signal detection without requiring external intervention or complex supporting infrastructure. This self-service capability reduces operational costs and increases accessibility.
2Productivity
If current sequencing platforms are used, then throughput is increased, but ceiling on number of possible reads per run limits further improvement
Solution Approach 1:
The patent divides the sequencing process into multiple independent microfluidic cartridges, each capable of performing a complete sequencing run. This segmentation allows parallel processing of multiple samples simultaneously, effectively removing the read capacity ceiling of single-instrument systems and enabling scalable throughput expansion.
Solution Approach 2:
The system transitions from single-instrument sequential processing to multi-cartridge parallel processing, adding a spatial dimension to throughput expansion. Multiple cartridges can be processed simultaneously in the same instrument or across multiple instruments, exponentially increasing total read capacity.
3Measurement precision
If current sequencing methods are used, then coverage is achieved, but biased coverage of GC-rich or AT-rich portions of genomes occurs
Solution Approach 1:
The patent employs multiple polymerases with different biochemical properties and optimal conditions, allowing adjustment of reaction parameters to sequence through difficult-to-sequence regions. By changing enzyme parameters and reaction conditions mid-run, the system achieves uniform coverage across GC-rich and AT-rich portions of genomes that would otherwise be poorly covered.
4Measurement precision
If current sequencing platforms are used, then accuracy is maintained, but inability to accurately sequence through homopolymer stretches occurs
Solution Approach 1:
The patent introduces intermediary structures including hairpin loops and blocking groups that mediate the sequencing reaction through homopolymer regions. These intermediaries allow the polymerase to navigate repetitive sequences by providing structural context and preventing slippage, thereby maintaining accuracy in homopolymer sequencing.
5Measurement precision
If current sequencing methods are used, then readability is achieved, but difficulty in sequencing beyond 200 nucleotides occurs resulting in difficulty in de novo assembly
Solution Approach 1:
The patent performs preliminary actions including template preparation with specific modifications and use of processive polymerases that are pre-optimized for long-read synthesis. These preliminary preparations enable the system to sequence beyond 200 nucleotides continuously, providing long reads that simplify de novo genome assembly without requiring complex computational scaffolding.
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 low-cost, rapid, and high-sensitivity DNA sequencing with improved accuracy and throughput, overcoming limitations of current platforms, particularly in sequencing RNA and challenging genomic regions.
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.
Implementation Method 2
fluorescence of the acceptor molecule is observed
Data Source
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 DNA.