Single Molecule Sequencing via Optical Labeling and Drift Correction
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Solution Overview
Problem
Second-generation sequencing technologies face issues with sequencing errors and biases in the PCR amplification process, which are not adequately addressed, and third-generation single molecule sequencing technologies, such as those developed by Helicos Corporation, have not gained market recognition due to high costs and other characteristics.
Innovation Solution
A single molecule sequencing method involving a template nucleic acid with optical detection labels at the 5' and/or 3' ends combined with a primer attached to a substrate, followed by imaging, polymerization with nucleotides having cleavable optical detection labels, and repeated cycles of extension and imaging to correct positional deviations and determine the nucleic acid sequence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If PCR amplification is used in second-generation sequencing, then sequencing throughput is improved, but sequencing errors and biases are introduced
Solution Approach 1:
The patent extracts and eliminates the PCR amplification step from the sequencing workflow. By using single-molecule sequencing technology, the method directly sequences individual DNA molecules without requiring prior amplification, thereby removing the source of amplification errors and biases while maintaining sequencing capability
Solution Approach 2:
The patent employs disposable single DNA molecules for sequencing instead of reusable amplified copies. Each DNA molecule is sequenced individually and then discarded, eliminating the need for PCR amplification and its associated errors while maintaining high throughput through parallel processing of multiple single molecules
2Reliability
If single molecule sequencing is implemented, then sequencing accuracy is improved, but cost increases
Solution Approach 1:
The patent uses optical copying/detection methods to read DNA sequences. By using fluorescently labeled nucleotides that emit light signals during incorporation, the system creates optical copies of the sequencing information that can be detected and recorded, enabling accurate sequencing without expensive physical manipulation of each molecule
Solution Approach 2:
The patent changes the detection parameter from requiring complex physical isolation and manipulation of single molecules to using fluorescent optical signals. By incorporating fluorescent labels on nucleotides and detecting their incorporation through light emission, the system reduces equipment complexity and operational costs while maintaining single-molecule sequencing accuracy
3Measurement precision
If positional drift correction is applied, then sequencing precision is improved, but processing time increases
Solution Approach 1:
The patent performs preliminary positioning of DNA molecules on the sequencing surface before the actual sequencing begins. By pre-aligning and fixing the positions of DNA molecules using imaging and coordinate recording, the system establishes a reference framework that minimizes the need for continuous correction during sequencing, thereby reducing processing time while maintaining precision
Solution Approach 2:
The patent implements a feedback mechanism where the positions of DNA molecules are continuously monitored and corrected during sequencing. By comparing actual positions against reference coordinates and applying real-time corrections to imaging or positioning systems, the method maintains high sequencing precision without requiring excessive processing time for correction
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 method achieves real-time single molecule sequencing with improved accuracy by correcting positional drift and enhancing sequencing precision through repeated extension reactions and image processing, potentially reducing costs and errors associated with earlier technologies.
Implementation Method 1
a template nucleic acid having a first optical detection label at the 5' and/or the 3' end of the template nucleic acid
Implementation Method 2
The company's academic paper, published in 2012, further improves the technology by directly sequencing individual DNA molecules with fluorescence
Data Source
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Figure 3(a)~3(b)
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AI summary
A sequencing method comprising: (i) combining a template nucleic acid having a first optical detection label at the end with a primer to obtain a first complex; (ii) imaging the first complex to obtain a first image; (iii) mixing the first complex, polymerase, and one or more of nucleotides with a optical detection label to obtain an extension product by polymerization reaction; (iv) imaging the extended first complex to obtain a second image; (v) removing the cleavable group of the nucleotides with the optical detection label from the extension product to obtain a second complex; (vi) repeating the above steps (ii) to (v) once or more times to determine the template nucleic acid sequence. The sequencing method can achieve single molecule sequencing by capturing template nucleic acids through primers.