Sequencing Probe Ligation for Signal Detection
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Solution Overview
Problem
Conventional sequencing methods are limited by signal degradation and low signal-to-noise ratios, restricting sequencing efficiency and making them unsuitable for single-molecule sequencing, while also requiring longer read lengths for accurate genomic analysis.
Innovation Solution
A method involving concatemers with adaptors and probes for hybridization and ligation, allowing for the detection of nucleotides through probe ligation products, enabling efficient sequencing and assembly of complete sequences from shorter read lengths.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional sequencing methods are used, then sequencing can be performed with simple methods, but signal degradation limits sequencing to only a few tens of nucleotides and low signal-to-noise ratios make single-molecule sequencing unsuitable
Solution Approach 1:
The sequencing probe is divided into distinct functional segments: a probe domain for target hybridization, a unique nucleotide at an interrogation position for base identification, and a label for detection. This segmentation allows each component to optimize its function, improving signal detection while maintaining manageable complexity
Solution Approach 2:
A unique nucleotide serves as an intermediary between the probe domain and the label. This intermediary element enables specific base identification through selective ligation while maintaining the structural integrity and detectability of the overall probe molecule
2Productivity
If conventional sequencing methods are used, then fewer sequencing steps are required, but read lengths are limited to a few tens of nucleotides due to signal degradation
Solution Approach 1:
The probe is pre-configured with the unique nucleotide at the interrogation position and the label attached before hybridization. This preliminary preparation ensures that when the probe hybridizes to the target, the detection components are already in place, enabling accurate base identification without signal degradation that would occur with longer conventional sequencing steps
Solution Approach 2:
The invention replaces conventional sequencing chemistry with a ligation-based detection system. Instead of relying on signal persistence through degradation-prone chemical reactions, the system uses specific enzymatic ligation of the probe to the target sequence, providing higher precision read lengths through a more stable biological mechanism
3Loss of time
If shorter read lengths are used for sequencing, then sequencing reactions can be completed faster, but more reads are required to assemble complete sequences
Solution Approach 1:
The label on the sequencing probe provides immediate feedback on successful hybridization and ligation events. This real-time detection allows for rapid confirmation of base identification, enabling faster sequencing reactions while maintaining the quality needed for efficient sequence assembly through high-confidence read data
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 efficiency and accuracy by improving signal detection and allowing for the assembly of complete sequences from shorter read lengths, overcoming the limitations of conventional methods.
Implementation Method 1
hybridizing at least a first sequencing probe to said first target domain, wherein said first sequencing probe comprises: i) a first probe domain complementary to said target domain
Implementation Method 2
ligating said anchor probes and said sequencing probe to form a probe ligation product
Data Source
Figure 1~3
Figure 2A~2B
Figure 2C
AI summary
The present invention is directed to compositions and methods for nucleic acid identification and detection. Compositions and methods of the present invention include extracting and fragmenting target nucleic acids from a sample, using the fragmented target nucleic acids to produce target nucleic acid templates and subjecting those target nucleic acid templates to amplification methods to form nucleic acid nanoballs. The invention also includes methods of detecting and identifying sequences using various sequencing applications, including sequencing by ligation methods.