Asymmetric Nucleic Acid Isolation via Hairpin Adapters
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Solution Overview
Problem
Current methods for isolating nucleic acid complexes are inefficient in distinguishing and isolating asymmetrically-primed templates, which are crucial for downstream analyses like sequence analysis, often resulting in incomplete or damaged templates that hinder sequencing quality.
Innovation Solution
The method involves obtaining a sample with both symmetrically- and asymmetrically-primed nucleic acid templates, using hairpin adapters and specific primers to immobilize symmetrically-primed templates on a solid support, and employing a strand-displacing polymerase to isolate asymmetrically-primed templates, ensuring they are synthesis-competent and free from damage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional nucleic acid isolation methods are used, then all nucleic acid templates are processed together, but asymmetrically-primed templates cannot be distinguished from symmetrically-primed templates, resulting in incomplete isolation and reduced sequencing quality
Solution Approach 1:
The patent applies asymmetry by designing asymmetric primers that bind to specific orientations of hairpin adapters on nucleic acid templates. The first primer binds to the 5' end hairpin adapter while the second primer binds to the 3' end hairpin adapter, creating an asymmetric binding pattern that distinguishes asymmetrically-primed templates from symmetrically-primed templates. This asymmetric design enables selective isolation of the desired template orientation.
Solution Approach 2:
The patent uses hairpin adapters as intermediary structures that facilitate selective primer binding. These adapters contain specific sequences that serve as binding sites for the asymmetric primers, acting as mediators between the nucleic acid templates and the isolation process. The hairpin structure allows for orientation-specific binding while maintaining template integrity.
2Reliability
If symmetrically-primed templates are not removed, then the isolation process is simpler, but damaged or incomplete templates are included in sequencing, reducing sequence analysis quality
Solution Approach 1:
The patent extracts or removes symmetrically-primed templates from the mixture through selective binding. By using asymmetric primers that only bind to asymmetrically-primed templates, the method effectively separates the desired templates from unwanted symmetrically-primed templates. The symmetrically-primed templates remain unbound and can be removed during washing steps, ensuring only high-quality templates proceed to sequencing.
Solution Approach 2:
The patent performs preliminary selection and validation of templates before sequencing. The asymmetric primer binding and subsequent amplification steps serve as preliminary actions that ensure only synthesis-competent, asymmetrically-primed templates are isolated. This preliminary filtering prevents damaged or incomplete templates from entering the sequencing process, improving overall reliability.
3Measurement precision
If asymmetric primers are used to isolate specific templates, then sequencing accuracy improves, but the isolation method becomes more complex requiring multiple primers and specific binding conditions
Solution Approach 1:
The patent segments the priming system into distinct functional components: a first asymmetric primer for binding to the 5' end hairpin adapter and a second asymmetric primer for binding to the 3' end hairpin adapter. This segmentation allows each primer to be optimized for its specific binding site and function, improving identification accuracy while organizing the complexity into manageable, modular components.
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 significantly improves the quality and length of sequencing reads by isolating only synthesis-competent, asymmetrically-primed templates, enhancing the accuracy and completeness of nucleic acid sequence analysis.
Implementation Method 1
wherein the capture oligonucleotide is specific for a capture region on the adapter
Implementation Method 2
contacting the nucleic acid templates immobilized to the solid support with a nucleic acid polymerase having strand displacement activity under conditions that promote nucleic acid synthesis from the synthesis primer
Data Source
AI summary
The present disclosure provides improved methods for isolating asymmetrically-primed and/or asymmetrically-tagged nucleic acid complexes that find use in downstream analytical analyses, including sequence analysis. Compositions comprising such complexes and kits and systems for generating such complexes are also provided.