Size-Controlled Nucleic Acid Fragments Using Active-Inactive Transposomes
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Solution Overview
Problem
Existing nucleic acid sequencing methods result in a broad size distribution of fragments, leading to inefficiencies in sequencing data generation and waste of nucleic acid samples, particularly in low concentration samples.
Innovation Solution
A transposome complex comprising a combination of active and inactive transposomes is used to generate size-controlled nucleic acid fragments by controlling the length of fragments through the ratio and arrangement of active and inactive transposomes, ensuring fragments are within the optimal size range for sequencing instruments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If mechanical fragmentation methods (sonicator, nebulizer, or nuclease) are used to generate DNA fragments, then DNA can be fragmented into smaller pieces, but the fragments exhibit a broad size distribution leading to waste of nucleic acid samples
Solution Approach 1:
The patent changes the key parameter of transposome composition by incorporating inactive transposomes alongside active transposomes. This parameter change enables precise control over fragment size distribution, producing a narrow size range that matches sequencing instrument requirements and reduces sample waste
Solution Approach 2:
Inactive transposomes serve as intermediaries that control the fragmentation process without catalyzing the reaction. They act as spatial regulators that define fragment boundaries, enabling size control while the active transposomes perform the actual fragmentation and tagging
2Ease of manufacture
If transposomes are used to tag and fragment DNA simultaneously, then library preparation is simplified, but the fragments still exhibit broad size distribution requiring additional size-selection steps
Solution Approach 1:
By adjusting the ratio and composition of active to inactive transposomes in the reaction mixture, the patent optimizes fragment size distribution to match sequencing instrument requirements, eliminating the need for additional size-selection steps while maintaining simplified library preparation
3Quantity of substance
If a broad size distribution of fragments is generated, then more nucleic acid material is available for sequencing, but sequencing efficiency decreases and data quality is reduced
Solution Approach 1:
The patent changes the fragmentation parameter from random mechanical shearing to controlled transposome-mediated cleavage with defined size distribution. This produces fragments within the optimal size range for sequencing instruments, maximizing sequencing efficiency and data quality while maintaining adequate material availability
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 reduces the amount of nucleic acid discarded and improves sequencing efficiency by generating fragments of uniform size, thereby enhancing data quality and reducing the need for additional size-selection steps.
Implementation Method 1
The use of transposomes, protein-DNA complexes of a transposase and transposon sequences that tag and fragment ('tagment') DNA by transposition
Implementation Method 2
hybridizing at least one linking transposome to the initiator transposome via an at least partially double-stranded linking adaptor
Data Source
AI summary
A transposome complex capable of producing size-controlled nucleic acid fragments is described herein. In some embodiments, the transposome complex includes multiple inactive transposomes with active transposomes on both ends of the multiple inactive transposomes. Applications, uses, and variations of the disclosed transposome complex include, but are not limited to, library preparation for a nucleic acid and tuning the length of the transposome complex to produce nucleic acid fragments of predetermined or desired lengths.