Split Mirrored Transposons for Nucleic Acid Contiguity Tracking
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Solution Overview
Problem
Current transpositional systems can introduce non-native sequences into target nucleic acids, but they lack the ability to track associations between fragmented and tagged nucleic acids, making it difficult to identify contiguity of nucleic acids after fragmentation.
Innovation Solution
The development of double-stranded transposon nucleic acid compositions that include restriction enzyme site sequences flanked by hairpin sequences, molecular identifier sequences, and mosaic end sequences, which can be complexed with transposase enzymes to generate a transposome complex, allowing for the insertion and tracking of associations between fragmented nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If transpositional systems are used to introduce non-native sequences into target nucleic acids, then sequence insertion capability is improved, but the ability to track associations between fragmented nucleic acids deteriorates
Solution Approach 1:
The transposon sequence is divided into multiple functional segments: molecular identifier sequences (MI sequences) that serve as tracking markers, hairpin sequences for structural stability, and mosaic end sequences for transposition activity. This segmentation allows the system to simultaneously perform sequence insertion while maintaining contiguity tracking through the MI sequences.
Solution Approach 2:
Molecular identifier sequences act as intermediary elements that bridge the gap between fragmentation and contiguity tracking. These MI sequences are inserted into the target nucleic acid along with the transposon, serving as detectable markers that preserve information about the original contiguity relationships even after the nucleic acid is fragmented.
2Productivity
If transposon sequences are used for fragmenting and tagging nucleic acids, then library generation efficiency is improved, but the ability to identify contiguity of nucleic acids deteriorates
Solution Approach 1:
The molecular identifier sequences function as informational 'color changes' or markers that can be detected through sequencing. These MI sequences provide distinct molecular signatures that allow precise identification of contiguity relationships between nucleic acid fragments, enabling accurate measurement of contiguity while maintaining high library generation efficiency.
3Adaptability or versatility
If additional sequences are appended to transposon sequences, then functionality is improved, but sequence complexity increases
Solution Approach 1:
The transposon sequence is designed as a multi-functional unit where: (1) mosaic end sequences provide transposition activity, (2) hairpin sequences provide structural stability and processing signals, and (3) molecular identifier sequences provide tracking capability. This universality allows a single transposon construct to perform multiple functions without proportionally increasing complexity, as each component serves a specific purpose.
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 enables the identification of contiguity of nucleic acids following fragmentation, allowing for the reassembly of contiguous nucleic acid sequences and enhancing the generation of nucleic acid libraries for applications like next-generation sequencing.
Implementation Method 1
Transpositional systems have been used successfully as a powerful tool for introducing non-native sequences into a target nucleic acid of interest. A transposome includes a transposase enzyme and transposon sequences, and the transposon sequences being specific to a particular transposase.
Implementation Method 2
a restriction enzyme site sequence flanked by first and second hairpin sequences
Data Source
AI summary
Disclosed herein are modified split mirrored transposons or double-stranded split mirrored transposon (SMT) compositions and methods of using SMTs. The methods include producing SMTs and using SMTs for nucleic acid analysis.