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
The method involves using nucleic acid constructs with adaptors and probes for hybridization and ligation, allowing for the detection of nucleotides through probe ligation products, enabling efficient sequencing even with shorter read lengths and improving signal detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional sequencing methods are used, then sequencing can be performed with standard techniques, but signal degradation limits the read length to only a few tens of nucleotides
Solution Approach 1:
The sequencing process is divided into multiple cycles, each determining a single nucleotide base. In each cycle, a probe with a specific terminal nucleotide is hybridized to the template, and only probes matching the template sequence are ligated. This segmented approach allows accurate determination of each base position independently, overcoming signal degradation that would affect longer reads taken as a single unit.
Solution Approach 2:
Before determining each nucleotide, the method performs preliminary hybridization of probes with specific terminal nucleotides to the template sequence. The probe hybridization step pre-positions the correct probe at the target location, ensuring that only probes with matching sequences are ligated in the subsequent step. This preliminary action enables accurate signal detection before degradation occurs.
2Reliability
If conventional sequencing methods are used, then standard sequencing protocols can be applied, but the signal-to-noise ratio is too low for single-molecule sequencing
Solution Approach 1:
The method combines hybridization and ligation into a single sequencing cycle. The probe must both hybridize to the template sequence and be ligated to the growing chain, merging two recognition steps into one. This combination increases the signal-to-noise ratio because both hybridization specificity and ligation efficiency must be satisfied simultaneously, reducing background noise while maintaining high sequencing efficiency through parallel processing of multiple templates.
Solution Approach 2:
The probe acts as an intermediary molecule that mediates between the template sequence and the detection system. The probe contains a terminal nucleotide that serves as a signal carrier, transferring sequence information from the template to the detection apparatus. This intermediary approach amplifies the signal by using the probe's labeled terminal nucleotide as a detectable marker, improving signal-to-noise ratio while enabling high-throughput sequencing of multiple molecules simultaneously.
3Loss of information
If longer read lengths are required for accurate genomic analysis, then more sequence information can be obtained per read, but conventional methods cannot achieve this due to signal degradation
Solution Approach 1:
The sequencing method maintains continuous useful action by performing multiple sequential cycles of probe hybridization and ligation. Each cycle determines one nucleotide and extends the sequenced region, continuously accumulating sequence information without interruption. This continuous process allows long read lengths to be achieved by maintaining signal quality throughout the sequencing process, with each cycle regenerating fresh probes and signals rather than relying on a single degrading signal.
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 allowing for the identification of nucleotides in a high-throughput manner, overcoming the limitations of conventional methods and enabling the assembly of complete sequences from shorter read lengths.
Implementation Method 1
hybridizing 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.