Restriction-Cleaved Single Nucleotide Probes for Faster Sequencing
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Solution Overview
Problem
Existing nucleic acid sequencing methods face limitations in signal generation rate and signal-to-noise ratio due to the speed of exonucleolytic digestion, which hampers accurate and rapid sequencing, especially for long reads.
Innovation Solution
A method involving the combination of exonucleolytic digestion with selective endonucleolytic cleavage of quenched fluorophore-bearing oligonucleotides, using probes with exonuclease blocking-sites and restriction endonuclease recognition-sites to enhance fluorescence signal generation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If exonucleolytic digestion is used to generate fluorescence signal, then nucleotide detection can be achieved, but the signal generation rate is limited by the speed of exonucleolytic digestion
Solution Approach 1:
The invention divides the signal generation process into two independent stages: (1) exonucleolytic digestion to release fluorophores from the 3' end of the probe, and (2) selective endonucleolytic cleavage at a restriction site to release additional fluorophores. This segmentation allows the slower exonucleolytic step to be complemented by a faster endonucleolytic step, thereby increasing the overall signal generation rate without compromising detection accuracy.
Solution Approach 2:
The invention introduces a restriction endonuclease as an intermediary enzyme that acts on a specific recognition site within the probe sequence. This intermediary enzyme provides an additional pathway for fluorophore release that is independent of the exonucleolytic digestion speed, thus mediating an increase in signal generation rate while maintaining the specificity required for accurate nucleotide detection.
2Measurement precision
If exonucleolytic digestion is used for nucleotide detection, then sequencing can be performed, but the signal-to-noise ratio is insufficient for rapid and accurate sequencing
Solution Approach 1:
The fluorescence signal generation is segmented into multiple sources: fluorophores released by exonucleolytic digestion and additional fluorophores released by selective endonucleolytic cleavage at restriction sites. This multi-source signal generation increases the overall signal intensity and improves the signal-to-noise ratio, enabling faster sequencing without sacrificing accuracy.
Solution Approach 2:
The invention changes the kinetic parameters of signal generation by introducing a second enzymatic step (endonucleolytic cleavage) with different rate characteristics. This parameter change accelerates the overall signal generation kinetics and enhances signal intensity, thereby improving the signal-to-noise ratio and reducing the time required for accurate nucleotide detection.
3Productivity
If faster sequencing is achieved by accelerating exonucleolytic digestion, then sequencing speed improves, but accuracy and reliability decrease
Solution Approach 1:
The invention segments the signal generation mechanism into exonucleolytic digestion (providing baseline signal) and selective endonucleolytic cleavage (providening enhanced signal). This segmentation allows the system to maintain high sequencing speed through the fast endonucleolytic step while preserving accuracy through the specificity of both enzymatic reactions. The dual-mechanism approach ensures that signal generation is both rapid and reliable.
Solution Approach 2:
The restriction endonuclease acts as an intermediary that provides a controlled, specific pathway for signal generation. Its activity at a defined recognition site ensures high fidelity signal release, complementing the exonucleolytic step and enhancing both speed and reliability of the sequencing process.
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 accelerates fluorescence signal growth, improving sequencing speed and accuracy by enhancing the signal-to-noise ratio, enabling faster and more reliable nucleotide detection.
Implementation Method 1
liberating the fluorophores from the quenchers and/or each other enabling them to fluoresce freely
Data Source
AI summary
A method of sequencing a nucleic acid comprising (1) generating a stream of single nucleoside triphosphates by progressive enzymatic digestion of the nucleic acid; (2) producing at least one oligonucleotide used probe by reacting, in the presence of a polymerase, at least one of the single nucleoside triphosphates with a corresponding biological probe comprising (a) a first single-stranded oligonucleotide including an exonuclease blocking-site, a restriction endonuclease recognition-site located on the 5′ side of the blocking-site and including a single nucleotide capture-site e, and at least one fluorophore region and (b) a second and optionally a third single-stranded oligonucleotide each separate from the first oligonucleotide; (3) cleaving the first oligonucleotide strand of the used probe at the recognition-site with a restriction endonuclease; (4) digesting the first oligonucleotide component with an enzyme to yield fluorophores in a detectable state and (5) detecting the fluorophores released in step (4).