Single Nucleotide Detection via Cyclic Fluorophore Release
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Solution Overview
Problem
Current DNA and RNA sequencing methods face challenges in reliably detecting single nucleotides due to weak fluorescence signals, which are often obscured by background noise, leading to reduced sensitivity and longer sequencing times.
Innovation Solution
A method involving progressive pyrophosphorolysis to generate a stream of single nucleoside triphosphates, which are then reacted with a probe system comprising labeled and unlabeled oligonucleotides. The probe system includes fluorophores that are initially quenched but become detectable upon capturing a nucleotide, allowing for enhanced fluorescence signals through exonucleolytic digestion and cyclic regeneration.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional sequencing methods are used, then sequencing can be performed, but the fluorescence signal is too weak to be reliably detected above background noise
Solution Approach 1:
The probe is divided into multiple fluorophores (at least two) attached to the same oligonucleotide, which are released and amplified through cyclic exonucleolytic digestion and probe regeneration. This segmentation and multiplication of fluorophores transforms a single weak signal into multiple amplified signals, enabling reliable detection above background noise
Solution Approach 2:
The patent implements a cyclic process where used probes are digested by exonucleases to release fluorophores, and the resulting oligonucleotides are recovered and reused to generate new probes. This recycling process continuously amplifies the fluorescence signal from each captured nucleotide, dramatically improving signal-to-noise ratio
2Measurement precision
If conventional sequencing methods are used, then sequencing can be performed, but sequencing time is extended due to weak signal detection
Solution Approach 1:
The patent establishes a continuous cyclic process where probes capture nucleotides, are digested to release fluorophores, and the oligonucleotide fragments are immediately reused to regenerate new probes. This continuous regeneration and reuse eliminates idle time between detection cycles, dramatically reducing total sequencing time while maintaining high detection accuracy
Solution Approach 2:
The sequencing process employs periodic cyclic actions of probe digestion, fluorophore release, and probe regeneration. Each cycle rapidly completes detection and prepares for the next nucleotide, creating an efficient rhythmic process that reduces overall sequencing time compared to conventional linear methods
3Reliability
If more fluorophores are generated per nucleotide, then detection sensitivity improves, but probe system complexity increases
Solution Approach 1:
The probe system is designed to be self-regenerating through automated exonucleolytic digestion and reuse of oligonucleotide fragments. Each captured probe automatically undergoes digestion to release fluorophores, and the resulting fragments are immediately reused to create new probes, eliminating the need for external intervention and simplifying system operation despite the cyclic complexity
Solution Approach 2:
The oligonucleotide fragments released from probe digestion serve multiple functions: they are both the byproduct of fluorophore release and the raw material for regenerating new probes. This multi-functionality allows the same molecular components to perform multiple roles in the cyclic process, reducing the need for additional separate reagents or system components
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 significantly improves the sensitivity of nucleic acid sequencing by generating more detectable fluorophores, making it easier to distinguish fluorescence signals above background noise, thereby shortening sequencing times and simplifying detection instrumentation.
Implementation Method 1
The probe system includes fluorophores that are initially quenched but become detectable upon capturing a nucleotide, allowing for enhanced fluorescence signals
Implementation Method 2
fluorophores that are initially quenched but become detectable upon capturing a nucleotide
Implementation Method 3
each of which can be caused to undergo exonucleolytic digestion to yield the fluorophores and a digestion product
Implementation Method 4
generating a stream of single nucleoside triphosphates by progressive pyrophosphorolysis of the nucleic acid
Data Source
AI summary
A method of analysing a single nucleoside triphosphate comprising: (1) producing at least one substantially double-stranded oligonucleotide used probe by reacting in the presence of a polymerase and a ligase the single nucleoside triphosphate with a corresponding probe system comprising (a) a first single-stranded oligonucleotide labelled with detectable elements in an undetectable state and (b) second and third single-stranded oligonucleotides capable of hybridising to complementary regions on the first oligonucleotide; (2) digesting the used probe with an enzyme having double-stranded exonucleolytic activity to yield the detectable elements in a detectable state and a single-stranded fourth oligonucleotide which is at least in part the sequence complement of the first oligonucleotide; (3) reacting the fourth oligonucleotide with another first oligonucleotide to produce a substantially double-stranded oligonucleotide product corresponding to the used probe; (4) repeating steps (2) and (3) in a cycle and (5) detecting the detectable elements released in each iteration.