Nucleic Acid Sequencing via Anchor Probe Hybridization
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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 costly and error-prone chained sequencing reactions.
Innovation Solution
The development of methods and compositions for sequencing reactions involving the use of adaptors, anchor probes, and sequencing probes to determine nucleotide sequences through hybridization and ligation, enabling efficient sequencing of nucleic acids with improved signal detection and error-free base reads.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional sequencing methods are used, then sequencing can be performed with existing technology, but signal degradation limits the read length to only a few tens of nucleotides
Solution Approach 1:
The invention segments the sequencing process into multiple independent cycles, where each cycle determines a single base position. By using separate anchor probes and sequencing probes for each position, the method achieves accurate determination of many consecutive bases without signal degradation, as each cycle starts with fresh probes rather than relying on cumulative signals from previous cycles
Solution Approach 2:
The invention performs preliminary hybridization of anchor probes to the template before sequencing begins. This preliminary action establishes stable binding sites that remain throughout the sequencing process, allowing multiple sequencing probes to bind and be detected without the signals degrading over time, thereby extending readable length
2Reliability
If conventional sequencing methods are used, then sequencing reactions can proceed with standard protocols, but signal-to-noise ratios are too low for single-molecule sequencing
Solution Approach 1:
The invention merges multiple detection functions into a single sequencing cycle. By combining the anchor probe binding function with the sequencing probe detection function, and using fluorescently labeled nucleotides that emit distinct signals for different bases, the method achieves high signal-to-noise ratios that enable reliable single-molecule sequencing
Solution Approach 2:
The invention uses fluorescently labeled nucleotides with different emission colors to represent different bases. This color-based encoding system provides strong, distinguishable signals that dramatically improve the signal-to-noise ratio, making single-molecule sequencing detectable and reliable
3Productivity
If chained sequencing reactions are used, then sequence determination can proceed through multiple cycles, but the process becomes costly and error-prone
Solution Approach 1:
The invention creates multiple copies of the template-bound anchor probe complex, each ready for independent sequencing probe binding. This copying approach allows parallel processing of multiple sequences simultaneously, increasing throughput while maintaining accuracy through redundant verification rather than sequential chaining that propagates errors
Solution Approach 2:
The invention incorporates detection and verification steps after each base determination. By detecting the incorporated nucleotide's fluorescent signal and using this feedback to inform the next sequencing cycle, the method achieves high accuracy without requiring error-prone chained reactions, as each cycle's result is verified before proceeding
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, reduces costs, and achieves high-throughput sequencing with accurate base reads and variant detection, suitable for genome association studies and clinical diagnostics.
Implementation Method 1
hybridizing an anchor probe to the anchor site
Implementation Method 2
hybridizing a pool of sequencing probes for determination of the sequence
Implementation Method 3
ligating the anchor probe and the sequencing probe
Data Source
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.