Form-Specific Nucleic Acid Processing for Liquid Biopsy Sensitivity
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Solution Overview
Problem
Existing liquid biopsy assays for cancer detection face challenges in sensitivity due to low amounts and heterogeneity of nucleic acids in body fluids, such as RNA and DNA, which are often lost during processing.
Innovation Solution
A method involving linking different forms of nucleic acids (e.g., double-stranded DNA, single-stranded DNA, single-stranded RNA) with specific tags, amplifying tagged nucleic acids, and assaying sequence data to decode the original template, while enriching for specific forms and modifications like 5-methylcytosine, using agents like methyl-binding domains to separate and tag nucleic acids based on modifications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Difficulty of detecting and measuring
If nucleic acids are processed and analyzed in liquid biopsy assays, then cancer detection capability is improved, but sensitivity is reduced due to low amounts and heterogeneity of nucleic acids
Solution Approach 1:
The patent segments the heterogeneous nucleic acid population into distinct forms (single-stranded DNA, double-stranded DNA, single-stranded RNA) and processes each form separately through form-specific enrichment and tagging. This segmentation allows optimized detection for each nucleic acid form, overcoming the sensitivity limitations imposed by processing all forms together as a heterogeneous mixture.
Solution Approach 2:
The patent applies local quality by implementing form-specific enrichment strategies for different nucleic acid forms (e.g., RNA-specific capture, ssDNA-specific capture) and using different tagging methods for different forms. This localized optimization ensures that each nucleic acid form is processed with the appropriate specificity, thereby improving overall detection sensitivity.
2Measurement precision
If nucleic acids are processed through enrichment and tagging steps, then sensitivity is improved, but loss of circulating nucleic acid increases
Solution Approach 1:
The patent performs preliminary actions by adding tags to nucleic acid molecules before enrichment and amplification steps. This preliminary tagging ensures that nucleic acids are identified and tracked throughout the multi-step process, allowing for efficient selection and reducing the risk of loss during subsequent enrichment operations.
Solution Approach 2:
The patent uses nucleic acid tags as intermediary molecules that facilitate the enrichment process. These tags serve as mediators between the nucleic acid molecules and the enrichment reagents, enabling specific capture and selection while minimizing direct interaction that could lead to nucleic acid degradation or loss.
3Device complexity
If all forms of nucleic acid are processed together, then workflow complexity is reduced, but measurement precision deteriorates due to heterogeneity
Solution Approach 1:
The patent segments the processing workflow into separate modules for different nucleic acid forms, with each module optimized for specific form characteristics. This segmentation, while increasing workflow complexity, enables precise measurement by eliminating the confounding effects of heterogeneity that would exist in a unified processing approach.
Solution Approach 2:
The patent employs universal tagging mechanisms that can be applied across different nucleic acid forms, with tags containing universal sequencing sites that work with standard sequencing platforms. This universality reduces the need for completely separate workflows for each form, thereby moderating the increase in workflow complexity while maintaining measurement precision.
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, allowing for the detection of somatic variants, copy number variations, and other genetic markers, improving cancer detection from bodily fluids.
Implementation Method 1
using agents like methyl-binding domains to separate and tag nucleic acids based on modifications
Implementation Method 2
linking at least one of the forms of nucleic acid with at least one tag nucleic acid
Implementation Method 3
the method further 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 4
amplifying the forms of nucleic acid at least one of which is linked to at least one nucleic acid tag
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.