Nucleic Acid Fragmentation for Uniform Capture
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Solution Overview
Problem
Current nucleic acid capture techniques face inefficiencies due to the poor uniformity of capture reactions, particularly with high molecular weight genomic nucleic acids, leading to inadequate coverage and increased costs and time in sequencing.
Innovation Solution
Fragmenting nucleic acids prior to capture reactions, such as using mechanical, chemical, or enzymatic methods, to expose target sites for capture moieties, thereby improving the uniformity and efficiency of capture reactions, especially with molecular inversion probes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If capture reactions are performed on whole genomic nucleic acids, then the complete genome can be analyzed, but the uniformity of capture is poor due to folding and melting temperature issues
Solution Approach 1:
The patent applies segmentation by fragmenting the long genomic nucleic acid into smaller fragments before performing capture reactions. This division into smaller segments resolves the folding and melting temperature problems that plague whole-genome capture, enabling uniform hybridization across all targets while maintaining coverage of the entire genome.
2Quantity of substance
If high molecular weight genomic nucleic acids are used, then comprehensive genomic coverage is achieved, but target exposure to capture moieties is prohibited due to natural folding
Solution Approach 1:
The patent applies preliminary action by performing fragmentation and denaturation steps before the capture reaction. This preliminary processing exposes the target sites on genomic nucleic acids by breaking down the folded structure, thereby enabling subsequent capture moieties to access and hybridize to targets effectively while maintaining comprehensive genomic coverage.
3Device complexity
If capture reactions are performed without fragmenting nucleic acid, then the process is simpler, but the distribution of abundances spans multiple orders of magnitude
Solution Approach 1:
The patent applies parameter changes by modifying the physical state of the nucleic acid through fragmentation and denaturation. These parameter changes transform the nucleic acid from a folded, inaccessible state to an exposed, hybridizable state, resulting in uniform target abundance distribution across the captured library without significantly increasing process complexity.
4Measurement precision
If a large number of sequencing reactions are performed to achieve effective coverage, then adequate target coverage is obtained, but costs and time to results increase
Solution Approach 1:
The patent applies mechanics substitution by replacing the mechanical approach of performing multiple sequencing reactions with a chemical/biochemical solution. By using fragmentation and denaturation to achieve uniform capture, the method obtains adequate target coverage in a single sequencing run, thereby reducing both time and costs associated with multiple sequencing reactions.
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 results in more uniform target abundance distribution, reducing failed captures and increasing the percentage of hybridized capture moieties, making the process more suitable for clinical diagnostics and reducing sequencing costs and time.
Implementation Method 1
capture moieties introduced to the genomic nucleic acid from being exposed to the targets for hybridization
Data Source
AI summary
The invention generally relates to methods of performing a capture reaction. In certain embodiments, the method involves obtaining a nucleic acid, fragmenting the nucleic acid, and capturing a target sequence on the nucleic acid fragment using a capture moiety, such as a molecular inversion probe.