Tagged Nucleic Acid Analysis for Sensitive Liquid Biopsy Sequencing
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Solution Overview
Problem
Existing liquid biopsy assays for cancer detection face challenges in sensitivity due to low amounts of heterogeneous nucleic acids in body fluids, such as RNA and DNA, which are often lost or not effectively analyzed.
Innovation Solution
A method involving linking different forms of nucleic acids (e.g., double-stranded DNA, single-stranded DNA, and single-stranded RNA) with specific tags, amplifying tagged nucleic acids, and decoding tag nucleic acid molecules to reveal the original template forms, allowing for enriched and sequenced analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If liquid biopsy assays are performed on body fluids to detect cancer, then noninvasive detection is achieved, but sensitivity is reduced due to low amounts of heterogeneous nucleic acids
Solution Approach 1:
The patent segments the heterogeneous nucleic acid population into distinct subsets based on form (double-stranded DNA, single-stranded DNA, RNA) by selectively amplifying each form separately. This segmentation allows for targeted enrichment and analysis of each nucleic acid type, overcoming the sensitivity limitation caused by low overall concentrations and heterogeneity in the original sample.
Solution Approach 2:
The patent applies preliminary selective amplification to enrich specific nucleic acid forms before analysis. By performing amplification steps that preferentially amplify certain nucleic acid forms (e.g., using form-specific primers or conditions), the method increases the concentration of target molecules prior to detection, thereby improving sensitivity while maintaining the noninvasive nature of the assay.
2Adaptability or versatility
If heterogeneous nucleic acid forms are analyzed together, then comprehensive cancer detection is achieved, but loss of circulating nucleic acid increases
Solution Approach 1:
The patent divides the analysis into separate amplification and detection steps for different nucleic acid forms. By processing double-stranded DNA, single-stranded DNA, and RNA separately through form-specific amplification protocols, the method reduces unnecessary manipulation and handling of each fragile nucleic acid type, thereby minimizing loss while maintaining comprehensive detection capability.
Solution Approach 2:
The patent introduces form-specific amplification intermediaries (such as form-specific primers, adapters, or enrichment steps) that enable selective amplification of each nucleic acid form. These intermediaries act as mediators that allow comprehensive analysis of all nucleic acid types while reducing direct handling and potential loss of the original circulating nucleic acids.
3Measurement precision
If selective amplification of different nucleic acid forms is performed, then sensitivity is improved, but device complexity increases
Solution Approach 1:
The patent employs universal amplification systems (such as PCR or isothermal amplification platforms) that can be configured for different nucleic acid forms through interchangeable primers, probes, or reaction conditions. This multi-functionality allows a single assay platform to handle multiple nucleic acid forms selectively, improving sensitivity without proportionally increasing device or system 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
Enhances the sensitivity and specificity of nucleic acid analysis in liquid biopsies, enabling detection of somatic variants, copy number variations, and other genetic markers with improved accuracy.
Implementation Method 1
step (a) comprises: subjecting the population to reverse transcription with a tagged primer, wherein the tagged primer is incorporated into cDNA generated from RNA in the population
Implementation Method 2
differentiating between the forms by ligating nucleic acid tags to the double-stranded DNA molecules
Implementation Method 3
The single-stranded DNA is separated by hybridization to one or more capture probes
Implementation Method 4
step (b) amplifying the forms of nucleic acid at least one of which is linked to at least one nucleic acid tag, wherein the nucleic acids and linked nucleic acid tag, if present, are amplified
Data Source
AI summary
The disclosure provides methods for processing nucleic acid populations containing different forms (e.g., RNA and DNA, single-stranded or double-stranded) and/or extents of modification (e.g., cytosine methylation, association with proteins). These methods accommodate multiple forms and/or modifications of nucleic acid in a sample, such that sequence information can be obtained for multiple forms. The methods also preserve the identity of multiple forms or modified states through processing and analysis, such that analysis of sequence can be combined with epigenetic analysis.