Circularized Nucleic Acid Analysis for Cancer Diagnosis
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Solution Overview
Problem
Current methods for analyzing nucleic acids from cell-free biological samples are limited in their ability to accurately diagnose diseases, particularly cancer, due to inefficiencies in processing and identifying specific nucleic acid molecules, especially when dealing with small sample quantities and varying methylation states.
Innovation Solution
The method involves bringing nucleic acid molecules into contact with binding agents, separating and circularizing them, followed by nucleic acid amplification and sequencing to identify specific sequences and methylation states, which can be processed against reference databases to determine disease presence or risk.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional nucleic acid analysis methods are used on cell-free samples, then the analysis can be performed with standard protocols, but the accuracy in diagnosing diseases is limited due to inefficiencies in processing and identifying specific nucleic acid molecules
Solution Approach 1:
The method segments the nucleic acid analysis process into distinct stages: (a) selective enrichment of target nucleic acids using binding agents specific to disease-associated sequences or modifications, (b) circularization of enriched nucleic acids to create unique topological structures, and (c) sequence-specific amplification. This segmentation allows each step to be optimized independently, improving both diagnostic accuracy and processing efficiency.
Solution Approach 2:
The patent introduces circularization as an intermediary step between enrichment and amplification. This intermediate transformation creates a topological signature that enhances the specificity of subsequent amplification and sequencing steps, thereby improving measurement precision without significantly increasing overall processing time.
2Measurement precision
If standard nucleic acid processing is applied to small sample quantities, then the sample can be processed without special handling, but the identification of specific nucleic acid molecules becomes less accurate
Solution Approach 1:
The method performs preliminary enrichment of target nucleic acid molecules using binding agents before the main analysis steps. This preliminary concentration of target molecules from small samples ensures sufficient quantity and purity for accurate identification, even when starting with limited biological material.
Solution Approach 2:
The patent changes the topological parameter of nucleic acids by circularizing them after enrichment. This parameter change creates a distinct structural state that enhances detection sensitivity and specificity, allowing accurate identification of molecules from small sample quantities that would be indistinguishable in linear form.
3Measurement precision
If nucleic acids are circularized after separation, then the circularized molecules can be amplified and identified with higher precision, but the overall process time increases
Solution Approach 1:
The method merges the circularization step with the amplification process by designing primers that can directly amplify circularized molecules. This integration eliminates separate purification and handling steps, reducing overall process time while maintaining the precision benefits of circularization.
Solution Approach 2:
The protocol maintains continuous useful action by performing circularization in a manner that allows immediate subsequent amplification without interruptive purification steps. The binding agents and circularization reagents are designed to be compatible with direct amplification, ensuring the useful analytical action continues uninterrupted.
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 precise diagnosis and monitoring of diseases like cancer by effectively processing small sample quantities, providing accurate identification of nucleic acid sequences and methylation states, thereby facilitating targeted therapeutic interventions.
Implementation Method 1
each of which specifically hybridizes to a different target sequence via sequence complementarity
Implementation Method 2
the binding agent comprises an antibody, or fragment thereof. In some cases, the antibody, or fragment thereof specifically binds to a nucleic acid binding protein
Implementation Method 3
the nucleic acid amplification is effected by a polymerase having strand-displacement activity
Implementation Method 4
the nucleic acid amplification is effected by a polymerase having strand-displacement activity
Data Source
AI summary
Provided herein are methods for processing a plurality of nucleic acid molecules derived from a cell-free biological sample, comprising bringing said plurality of nucleic acid molecules or derivatives thereof in contact with a plurality of binding agents, to provide a first subset of said plurality of nucleic acid molecules coupled to said plurality of binding agents and a second subset of said plurality of nucleic acid molecules; separating said first subset of said plurality of nucleic acid molecules coupled to said plurality of binding agents from said second subset of said plurality of nucleic acid molecules; circularizing a nucleic acid molecule derived from said first subset of said plurality of nucleic acid molecules to obtain a circularized nucleic acid molecule; and identifying said circularized nucleic acid molecule or derivative thereof.