Rolling Circle Amplification of Dumbbell DNA Templates
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current whole genome sequencing methods, particularly next-generation sequencing (NGS), face limitations in amplifying large DNA fragments and resolving structural variants due to size constraints and complexity, leading to incomplete characterization of genomic sequences and biased understanding of disease mechanisms.
Innovation Solution
The method involves creating dumbbell templates by fragmenting DNA, ligating hairpin structures to the ends, and performing rolling circle replication or amplification using substantially complementary primers attached to a substrate, allowing for size-independent replication or amplification of large DNA circles, overcoming the limitations of existing methods.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of moving object
If current whole genome sequencing methods are used, then sequencing can be performed, but large DNA fragments cannot be amplified effectively due to size constraints
Solution Approach 1:
The DNA molecule is divided into smaller fragments that can be individually processed and amplified. The system segments large genomic DNA into manageable pieces that fit within the amplification capabilities of the rolling circle system, allowing comprehensive genome coverage while maintaining amplification efficiency
Solution Approach 2:
Hairpin adapter structures serve as intermediaries between the large DNA fragments and the rolling circle amplification system. These hairpin adapters act as mediators that enable the amplification machinery to process large DNA molecules by providing appropriate binding sites and structural frameworks
2Loss of information
If large DNA fragments are sequenced, then complete genomic information is obtained, but the complexity of the analysis increases significantly
Solution Approach 1:
The genome is segmented into multiple large fragments that are independently amplified and sequenced. This segmentation allows the complex genomic information to be processed in manageable units while maintaining complete genome coverage, reducing the overall analytical complexity
Solution Approach 2:
The system adds dimensional organization through the use of hairpin adapters and rolling circle replication structures. This creates a structured, multi-dimensional framework for organizing and analyzing genomic data, transforming complex linear sequences into organized circular templates
3Measurement precision
If structural variants are resolved, then accurate genomic characterization is achieved, but the requirement for large DNA fragment amplification increases system demands
Solution Approach 1:
Hairpin adapters serve as intermediaries that facilitate the detection and resolution of structural variants. These adapters provide standardized binding sites and structural markers that enable precise identification of structural variations without requiring excessively complex system configurations
Solution Approach 2:
The system performs preliminary amplification of large DNA fragments using rolling circle replication before sequencing. This preliminary action ensures that sufficient quantities of large DNA fragments are available for structural variant analysis, reducing the need for complex post-processing and high-demand detection systems
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 efficient replication and amplification of large DNA templates, facilitating comprehensive genomic analysis, including de novo assembly of complex genomes, and providing detailed haplotype information, thereby improving the characterization of genomic sequences and understanding of disease mechanisms.
Implementation Method 1
performing rolling circle replication on the at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one replicated dumbbell template
Implementation Method 2
performing rolling circle amplification on the at least one dumbbell template contacted with the at least one substantially complementary primer to form at least one amplified DNA molecule
Implementation Method 3
ligating one or more hairpin structures to each end of the at least one fragmented DNA molecule
Data Source
AI summary
The present invention provides for methods, reagents, apparatuses, and systems for the replication or amplification of nucleic acid molecules from biological samples. In one embodiment of the invention, the nucleic molecules are isolated from the sample, and subjected to fragmenting and joining using ligating agents of one or more hairpin structures to each end of the fragmented nucleic molecules to form one or more dumbbell templates. The one or more dumbbell templates are contacted with at least one substantially complementary primer attached to a substrate, and subjected to rolling circle replication or rolling circle amplification. The resulting replicated dumbbell templates or amplified dumbbell templates are used in numerous genomic applications, including whole genome de novo sequencing; sequence variant detection, structural variant detection, determining the phase of molecular haplotypes, molecular counting for aneuploidy detection; targeted sequencing of gene panels, whole exome, or chromosomal regions for sequence variant detection, structural variant detection, determining the phase of molecular haplotypes and/or molecular counting for aneuploidy detection; study of nucleic acid-nucleic acid binding interactions, nucleic acid-protein binding interactions, and nucleic acid molecule expression arrays; and testing of the effects of small molecule inhibitors or activators or nucleic acid therapeutics.