Nucleic Acid Counting via Rolling Circle Amplification
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Solution Overview
Problem
Current methods for quantifying variations in numbers of molecules, such as those used in aneuploidy screening, are often time-consuming, expensive, and require extensive bioinformatics analysis, particularly relying on next generation sequencing (NGS) technologies.
Innovation Solution
The technology provides compositions, methods, and systems for detecting and characterizing samples by counting specific molecules, such as nucleic acids, without the use of NGS or single-molecule amplification technologies. This is achieved through rolling circle amplification (RCA) and the use of molecular inversion probes (MIPs), which allow for the detection and counting of single copies of target molecules.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If next generation sequencing (NGS) technologies are used for quantifying variations in numbers of molecules, then measurement precision is improved, but loss of time and device complexity increase
Solution Approach 1:
The patent divides the complex NGS process into separate functional modules: molecular inversion probes for target capture, rolling circle amplification for signal amplification, and imaging for detection. Each module performs a specific function independently, enabling parallel processing and reducing overall analysis time while maintaining quantification precision.
Solution Approach 2:
The patent performs preliminary target capture and enrichment using molecular inversion probes before amplification and detection. This preliminary action concentrates the target molecules of interest, allowing for faster and more efficient subsequent processing steps while maintaining high measurement precision.
2Measurement precision
If next generation sequencing (NGS) technologies are used for quantifying variations in numbers of molecules, then measurement precision is improved, but device complexity increases
Solution Approach 1:
The patent segments the NGS system into distinct functional components: molecular inversion probes for specific target capture, rolling circle amplification for signal amplification, and imaging systems for detection. This segmentation allows each component to be optimized independently and simplifies the overall system architecture.
Solution Approach 2:
The patent introduces molecular inversion probes as intermediary elements that specifically capture target molecules before amplification. These probes act as mediators between the complex NGS system and the target molecules, simplifying the interaction and reducing system complexity while maintaining measurement precision.
3Manufacturing precision
If single-molecule amplification technologies are used, then manufacturing precision is improved, but device complexity and loss of time increase
Solution Approach 1:
The patent merges target capture, amplification, and detection into a single integrated rolling circle amplification system. This merging eliminates the need for separate single-molecule amplification devices, reducing device complexity while maintaining manufacturing precision through the isothermal amplification process.
Solution Approach 2:
The rolling circle amplification system is self-amplifying, where the amplification process generates its own signal without requiring external intervention or complex instrumentation. The circular template and primer system automatically generate multiple copies of the target sequence, providing self-service amplification that reduces device complexity.
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 improved detection of genomic deletions and duplications of various sizes, including complete chromosomes, and allows for non-invasive prenatal testing (NIPT) and other applications without the need for extensive sequencing or amplification processes.
Implementation Method 1
extending primers in the complexes in a rolling circle amplification (RCA) reaction to form RCA products
Implementation Method 2
treating the ligation mixture with at least one exonuclease, wherein circularized nucleic acid probes are not substrate for the at least one exonuclease
Implementation Method 3
forming a plurality of complexes, each complex comprising an oligonucleotide primer hybridized to a circularized nucleic acid probe
Data Source
AI summary
Compositions and methods, systems, and kits for detecting and quantifying variations in numbers of molecules, particularly variations in gene dosage, e.g., due to gene duplication, or to variations from the normal euploid complement of chromosomes, e.g., trisomy of one or more chromosomes that are normally found in diploid pairs, without digital sequencing.