Nanowell Sequencing Apparatus with Immobilized Polymerase Complexes
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Solution Overview
Problem
Conventional single molecule sequencing methods require precise loading and maintenance of a single polymerase and nucleic acid template, leading to narrow operation windows and high failure rates due to polymerase activity loss or template release.
Innovation Solution
An apparatus with nanowells and immobilization moieties for nucleic acid fragments and polymerases, allowing simultaneous immobilization of multiple fragments and polymerases, with only one primer-polymerase-nucleic acid complex formed at a time, enabling continuous sequencing without re-loading and reducing sample preparation time.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If precise loading process is used to maintain one template and allow only one template in each detectable zone, then sequencing accuracy is improved, but operation window is narrowed and failure rate increases
Solution Approach 1:
The system divides the detectable zone into multiple nanowells, each capable of independently accommodating template-polymerase complexes. This segmentation allows parallel sequencing reactions while maintaining the precision of single-molecule detection in each nanowell, thereby improving overall reliability without sacrificing accuracy.
Solution Approach 2:
Multiple template-polymerase complexes are pre-loaded into the nanowells before the sequencing reaction begins. This preliminary action ensures that all necessary components are in place and properly positioned, eliminating the need for precise real-time loading during the sequencing process and expanding the operation window.
2Duration of action of moving object
If polymerase with strand displacement activity and high processivity is used, then sequencing continuity is improved, but system complexity increases due to strict activity requirements
Solution Approach 1:
Multiple copies of template-polymerase complexes are loaded into the nanowell array. If one complex loses activity or releases the template, other copies can continue the sequencing reaction, providing redundancy that maintains sequencing continuity without requiring extremely high processivity from each individual polymerase.
Solution Approach 2:
The system changes the parameter of polymerase quantity from single to multiple, and alters the operational mode from sequential to parallel processing. This allows the use of polymerases with moderate processivity requirements while maintaining overall sequencing continuity through the collective action of multiple complexes.
3Measurement precision
If single molecule sequencing-by-synthesis is performed with statistical consensus of useful redundant reads, then sequencing accuracy is improved, but sample preparation time increases
Solution Approach 1:
Multiple sequencing reactions are merged into a single observation zone containing multiple nanowells. The statistical consensus is obtained by combining signals from multiple template-polymerase complexes simultaneously, rather than performing separate sequencing reactions sequentially. This merging approach maintains accuracy while dramatically reducing sample preparation and processing time.
Solution Approach 2:
Multiple sequencing reactions proceed continuously and simultaneously in parallel within the same observation zone. Instead of completing one sequencing reaction before starting the next, the system maintains continuous useful action across multiple reactions, eliminating idle time and reducing overall preparation time while preserving accuracy through consensus sequencing.
Data Source
Figure 1A~1B
Figure 1C~1D
Figure 2
AI summary
An apparatus (100, 100a) suitable for single molecule sequencing. The apparatus (100, 100a) includes at least one nanowell (110), a plurality of nucleic acid immobilization moieties (120a, 120b, 120c, 120d, 120e), and a plurality of types of nucleic acid fragments (130a, 130b, 130c, 130d, 130e). The nanowell (110) has an observation zone (112). The nucleic acid immobilization moieties (120a, 120b, 120c, 120d, 120e) are disposed in or proximate to the observation zone (112). The nucleic acid fragments (130a, 130b, 130c, 130d, 130e) are immobilized to the nucleic acid immobilization moieties (120a, 120b, 120c, 120d, 120e), respectively. At least one polymerase (140a, 140b, 140c, 140d) is disposed in the observation zone (112). A method of sequencing nucleic acid molecules using the above-mentioned apparatus is provided.