Gapped Circle Nucleic Acid Template for Sequencing
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Solution Overview
Problem
Current nucleic acid sequencing methods require efficient and cost-effective means to form circular templates for accurate long-range sequencing, particularly for single molecule sequencing technologies like nanopore-based methods, where existing methods are either costly or inefficient.
Innovation Solution
A novel nucleic acid template structure known as a 'gapped circle' is introduced, comprising a double-stranded circle with a short single-stranded gap, created by introducing nicks in one strand using nicking enzymes or uracil glycosylase, allowing for sequencing initiation from an extendable 3′-end and enabling consensus sequencing through multiple reads.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If circular templates are used for single molecule sequencing, then sequencing accuracy is improved through consensus from multiple reads, but the cost and complexity of template formation increases
Solution Approach 1:
The circular template is segmented into a double-stranded circular portion and a single-stranded gap portion. This segmentation allows the template to be formed from linear DNA through targeted nicking and gap creation, simplifying the overall formation process while maintaining the circular topology needed for multiple reads and consensus sequencing.
Solution Approach 2:
Adaptors are used as intermediary elements to facilitate template formation. The adaptors contain cleavage sites that enable controlled nicking by nicking enzymes, and they provide primer binding sites that facilitate circularization. This intermediary approach simplifies the complex process of converting linear DNA to circular templates.
2Ease of manufacture
If nicks are introduced into one strand of double-stranded circle, then template formation is simplified, but the stability of the circular structure may be compromised
Solution Approach 1:
The template structure exhibits local quality differences: the double-stranded circular portion maintains full stability and continuity, while the single-stranded gap portion provides controlled flexibility and extendability. This localized variation allows the template to be stable enough for multiple reads while having a specific region that facilitates primer binding and sequencing initiation.
Solution Approach 2:
The gap is created as a preliminary feature during template formation, positioned specifically to accommodate primer binding sites. This preliminary action ensures that when the template is used for sequencing, the primer can bind at the correct location to initiate synthesis, while the rest of the circular structure remains stable and intact.
3Measurement precision
If multiple reads of the same molecule are performed, then consensus sequence determination is improved, but the time required for sequencing increases
Solution Approach 1:
The circular template structure enables continuous sequencing action by allowing the polymerase to read the same template molecule multiple times in succession. The circular topology permits the sequencing process to continue without removing the template, maintaining useful action throughout and enabling multiple reads from the same molecule to improve consensus accuracy.
Solution Approach 2:
The single-stranded gap is designed to be filled during the sequencing process itself. As the polymerase reads through the gap region, it synthesizes the complementary strand, effectively recovering the continuous double-stranded structure. This allows the template to be reused for subsequent reads without degradation.
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 gapped circle template structure enhances sequencing accuracy and efficiency by allowing multiple reads of the same molecule, reducing sequencing errors and improving consensus sequence determination, while being economically viable for high-throughput sequencing applications.
Implementation Method 1
The nicks are created by a nicking enzyme recognizing its specific binding sequence or by a glycosylase recognizing uracil bases in combination with a second enzyme forming a single-stranded break (nick)
Implementation Method 2
extending the extendable 3′-end to copy at least a portion of the circular strand
Data Source
AI summary
Disclosed is a novel structure of a nucleic acid template and the method of making and using the structure. The structure consists of a double-stranded circle with a single-stranded gap. The circular gapped structure includes an extendable end from which copying or sequencing can be initiated.