Circular DNA Adaptor Insertion via Segmented Recognition Sites
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Solution Overview
Problem
Current sequencing methods face challenges in achieving low-cost, high-throughput sequencing and re-sequencing of genomic DNA, particularly in protecting restriction endonuclease recognition sites and controlling the orientation and position of DNA adaptors, which limits sequence representation and data quality.
Innovation Solution
The method involves repeated cycles of nucleic acid cleavage and ligation to insert multiple DNA adaptors into circular target DNAs at defined positions and orientations, using the same Type IIS restriction endonuclease recognition site for all adaptors, and employing sequence-specific nickases to protect these sites, allowing for consecutive insertion and orientation control.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If restriction endonuclease recognition sites are used for adaptor insertion, then adaptor ligation efficiency is improved, but sequences around restriction sites are excised leading to loss of sequence representation
Solution Approach 1:
The adaptor is divided into two separate arms, each containing a recognition site half-site. These arms are ligated to different ends of the target DNA fragment, allowing the restriction enzyme to cut outside the fragment while maintaining high ligation efficiency at the adaptor-target junctions
Solution Approach 2:
A linker sequence is introduced as an intermediary between the restriction enzyme recognition site and the target DNA fragment. This linker allows the restriction enzyme to bind and cut at the recognition site while preventing excision of the target sequence, thereby preserving sequence representation
2Manufacturing precision
If multiple adaptors are inserted into target DNA, then sequencing data quality and quantity are improved, but control over adaptor position and orientation becomes more difficult
Solution Approach 1:
Different functional elements are placed at specific locations within the adaptor structure: recognition site half-sites are positioned at the ends for efficient ligation, while orientation-control sequences and indexing regions are placed in specific orientations and positions to enable precise control over adaptor placement and subsequent sequencing reads
Solution Approach 2:
The two adaptor arms are designed with asymmetric sequences and orientations. Each arm contains specific sequences that direct their insertion into the target DNA in a defined orientation, ensuring that multiple adaptors are inserted consistently with controlled positions and orientations relative to the target fragment
3Ease of manufacture
If the same restriction endonuclease recognition site is used in all adaptors, then process simplicity is improved, but protection of recognition sites from excision becomes more challenging
Solution Approach 1:
The complete recognition site is segmented into two half-sites distributed on separate adaptor arms. Each half-site alone is insufficient for restriction enzyme binding and cutting, providing inherent protection while maintaining the ability to use the same recognition site sequence design across all adaptors for process simplicity
Solution Approach 2:
The adaptor arms are designed with recognition site half-sites in a configuration that prevents excision before the adaptor is ligated to the target DNA. This preliminary design ensures that when the restriction enzyme is added, the recognition sites are already protected by the adaptor structure, preventing unwanted excision events
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 the quality and quantity of sequencing data by avoiding excision of sequences around restriction sites and enabling precise control over adaptor insertion, leading to improved sequence representation and data accuracy in massively parallel sequencing techniques.
Implementation Method 1
digesting the first library constructs with a restriction endonuclease that recognizes the restriction endonuclease recognition site in the first adaptor
Implementation Method 2
creating single-stranded regions in the first and second adaptor arms at the restriction endonuclease recognition site
Implementation Method 3
amplifying the first library constructs
Implementation Method 4
ligating a first arm and a second arm of a first adaptor to the target nucleic acids
Data Source
AI summary
Aspects described and claimed herein provide methods to insert multiple DNA adaptors into a population of circular target DNAs at defined positions and orientations with respect to one another. The resulting multi-adaptor constructs are then used in massively-parallel nucleic acid sequencing techniques.