Single-Molecule Sequencing With Cleavable Optical Labels
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Solution Overview
Problem
Single-molecule sequencing technologies face limitations in sequencing error rate, sequencing time, and sequencing read length, particularly in multi-color sequencing methods.
Innovation Solution
A single-molecule sequencing method involving a solid carrier with immobilized nucleic acid template-primer complexes, using four types of nucleotides or their analogs with different optically detectable labels, followed by optical signal generation, label removal, and repeated cycles to determine the nucleic acid sequence, optimizing conditions such as pH, temperature, and cleavage reagents to improve accuracy and efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multi-color sequencing is used to shorten sequencing time, then productivity is improved, but sequencing error rate increases and measurement precision deteriorates
Solution Approach 1:
The patent segments the multi-color sequencing process into distinct stages: (1) simultaneous addition of multiple nucleotide types with different optically detectable labels, (2) single-base extension reaction, (3) optical signal acquisition, and (4) label removal. This segmentation allows parallel processing of multiple nucleotides while maintaining single-base extension accuracy, thereby reducing sequencing time without increasing error rates.
Solution Approach 2:
The patent optimizes reaction parameters including nucleotide concentration ratios, polymerase activity, and optically detectable label characteristics to enable simultaneous addition of multiple nucleotide types while maintaining single-base extension fidelity. By carefully controlling these parameters, the system achieves both high productivity and low error rates in multi-color sequencing.
2Productivity
If multi-color sequencing is used to reduce sequencing time, then productivity is improved, but sequencing read length is limited
Solution Approach 1:
The patent implements continuous single-base extension reactions with optically detectable labels that can be sequentially imaged over multiple cycles. The optically detectable labels remain attached during imaging and are removed only after complete sequence reading, enabling continuous acquisition of sequence information and achieving both high productivity and long read lengths.
Solution Approach 2:
The optically detectable labels serve as intermediaries that bridge the simultaneous multi-nucleotide addition and sequential single-base extension requirements. These labels allow parallel nucleotide incorporation while enabling sequential detection through optical imaging, thus extending read length without sacrificing sequencing speed.
3Productivity
If four types of nucleotides are simultaneously added for single-base extension, then productivity is improved, but manufacturing precision deteriorates
Solution Approach 1:
The patent applies local quality by giving each nucleotide type distinct optically detectable labels with different spectral characteristics. This allows the system to differentiate between multiple simultaneously added nucleotide types at each binding site, maintaining single-base extension accuracy while enabling parallel processing of all four nucleotide types.
Solution Approach 2:
The patent uses optically detectable labels with different optical properties (colors) to distinguish between multiple nucleotide types added simultaneously. Each nucleotide type carries a unique optical signature, enabling accurate identification of incorporated bases through optical detection while maintaining high sequencing throughput.
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
The method reduces sequencing error rates, enhances sequencing quality, and shortens sequencing time while achieving longer read lengths compared to traditional methods.
Implementation Method 1
exciting the optically detectable label to generate an optical signal, and imaging the extension product to obtain an image
Data Source
AI summary
The present disclosure relates to the field of sequencing, and in particular to, a single-molecule sequencing method. The present disclosure provides the single-molecule sequencing method, comprising the following steps: (i) providing a solid carrier comprising a plurality of sites, each of the sites having a nucleic acid template-primer complex immobilized thereon; (ii) binding four types of nucleotides or analogs thereof carrying different optically detectable labels to the nucleic acid template-primer complex by a polymerization reaction to achieve single-base extension and obtain an extension product; (iii) exciting the optically detectable label to generate an optical signal, and imaging the extension product to obtain an image; (iv) removing the optically detectable label on the extension product; (v) (iii) replacing the nucleic acid template-primer complex with the extension product from which the optically detectable label is cleaved, and repeating (ii) to (iv) above one or more times to determine a template nucleic acid sequence. The single-molecule sequencing method provided by the present disclosure is beneficial to reducing the error rate, improving the sequencing quality, and shortening the sequencing time.