Tailing-Based Nucleic Acid Library Preparation for Rare Targets
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Solution Overview
Problem
Existing methods face challenges in detecting nucleic acid species present in low abundance due to small sample input and errors in manipulation, particularly in applications like environmental testing and disease detection.
Innovation Solution
A method involving template-independent polymerization to add tails to target polynucleotides, followed by adapter ligation and amplification, to enhance the preparation of nucleic acid libraries.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional nucleic acid library preparation methods are used, then the process is simple, but detection sensitivity for low-abundance nucleic acids is insufficient
Solution Approach 1:
The patent applies preliminary action by performing tailing reactions before ligation to add protective tails and adapters to target polynucleotides. This preliminary modification stabilizes low-abundance nucleic acids and prevents loss during subsequent processing steps, thereby improving detection sensitivity without requiring fundamental changes to the overall workflow
Solution Approach 2:
The patent uses adapter molecules as intermediaries between target polynucleotides and sequencing platforms. These adapters facilitate ligation and amplification while protecting rare nucleic acid species from degradation, enabling sensitive detection of low-abundance targets through multiple intermediate steps rather than direct processing
2Measurement precision
If sample input is kept small to maintain purity, then contamination is reduced, but detection of low-abundance nucleic acid species becomes difficult
Solution Approach 1:
The patent changes the chemical parameters of the sample by adding protective tails and adapter sequences to target polynucleotides. This modification transforms rare nucleic acid species into stabilized complexes that can be detected at lower concentrations, effectively improving detection capability without requiring increased sample input
Solution Approach 2:
The patent segments the sample processing into distinct stages: tailing, ligation, and amplification. Each stage is optimized to preserve low-abundance species, with the tailing step specifically designed to protect rare nucleic acids from loss during fragmentation and purification, enabling detection without large sample inputs
3Reliability
If manual manipulation steps are used to handle samples, then flexibility is maintained, but errors occur during processing
Solution Approach 1:
The patent implements self-service by designing tailing reactions that automatically protect target polynucleotides from degradation and loss. The protective tails and adapters self-assemble onto target molecules during the reaction, reducing the need for careful manual handling and minimizing human-induced errors without complicating the overall procedure
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
Improves the detection sensitivity of low-abundance nucleic acids by stabilizing and amplifying them effectively, facilitating accurate analysis.
Implementation Method 1
adding a first tail to each of a plurality of target polynucleotides by template-independent polymerization, wherein the first tailing reaction comprises a first adapter
Implementation Method 2
a first adapter comprising an overhang that hybridizes to the first tail
Implementation Method 3
in a first ligation reaction, ligating a strand of the first adapter to the first tail
Implementation Method 4
amplifying target polynucleotides comprising the strand of the first adapter by extending a first primer hybridized to the strand of the first adapter
Data Source
AI summary
In various aspects, the present disclosure provides methods, compositions, reaction mixtures, kits, and systems for preparing nucleic acid libraries, such as for polynucleotide sequencing. In some embodiments, preparation methods comprise tailing reactions, ligation reactions for attaching an adapter, and an amplification reaction between ligation reactions.