Long Nucleic Acid Isolation via Reducing Agents and Protease
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Solution Overview
Problem
Conventional methods for isolating nucleic acids, especially single-stranded RNA, result in breakage and chemical degradation, leading to recovery of short strands, which is inadequate for long-read next-generation sequencing applications that require nucleic acid molecules greater than 1,000bp.
Innovation Solution
A novel method involving a multi-step process using lysis buffers with reducing agents, detergents, proteases, and a nucleic acid-retaining separation matrix, along with DNase treatment and concentration via semipermeable membranes, to isolate and preserve long RNA molecules, including viral RNA, from various samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Length of moving object
If conventional nucleic acid isolation methods are used, then the isolation process is simple and quick, but the nucleic acid molecules break or degrade resulting in short strands that are inadequate for long-read sequencing
Solution Approach 1:
The patent modifies multiple parameters of the isolation process including using a two-step lysis buffer system with specific reducing agents (beta-mercaptoethanol, DTT) at controlled concentrations, adjusting incubation temperatures (55°C for protease digestion), and controlling elution conditions. These parameter changes collectively preserve nucleic acid integrity while maintaining recovery efficiency.
Solution Approach 2:
The patent introduces several intermediary substances to protect nucleic acids during isolation: reducing agents that prevent disulfide bond formation and degradation, proteases that digest proteins without damaging nucleic acids, and carrier molecules that stabilize the isolation process. These intermediaries create a protective environment for long nucleic acid strands.
2Measurement precision
If conventional isolation methods are used, then the process is fast and efficient, but the limit of detection is poor for low copy number viral RNA
Solution Approach 1:
The patent performs preliminary concentration of viral particles from large sample volumes (up to 100 mL) before nucleic acid extraction. This pre-concentration step enriches low copy number targets, enabling detection limits of 1-10 copies/mL while maintaining overall process efficiency through automated workflows.
Solution Approach 2:
The isolation process is divided into distinct modular steps: viral particle concentration, lysis with reducing agents, protease digestion, nucleic acid binding to silica matrix, washing, and elution. This segmentation allows optimization of each step for sensitivity while maintaining overall productivity through parallel processing options.
3Stability of the object's composition
If single-stranded RNA is isolated using traditional protocols, then the process is straightforward, but the RNA is easily degraded resulting in loss of long molecules
Solution Approach 1:
The patent maintains RNA stability by controlling temperature parameters (incubation at 55°C for protease, lower temperatures for RNA-sensitive steps), pH conditions in buffers, and adding stabilizing agents like RNase inhibitors. These parameter controls prevent degradation while keeping the protocol manageable through standardized buffer systems.
Solution Approach 2:
The patent uses protease K as an intermediary that digests proteins including RNases, thereby protecting RNA from degradation. Reducing agents serve as intermediaries that prevent oxidative damage to RNA. These intermediaries add steps to the protocol but are essential for maintaining RNA integrity throughout the isolation process.
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
The method significantly increases the sensitivity of nucleic acid recovery, achieving a 10-fold improvement in limit of detection and up to 5-fold improvement in yield of longer nucleic acid fragments, particularly from samples with low viral loads, enabling more effective long-read sequencing.
Implementation Method 1
contacting the sample with a first lysis buffer comprising a reducing agent
Implementation Method 2
contacting the sample with a first lysis buffer comprising a reducing agent, a detergent
Implementation Method 3
contacting the sample with a second lysis buffer containing a protease at a temperature between 20-55°C
Implementation Method 4
contacting the sample with a second lysis buffer containing a protease
Implementation Method 5
contacting the sample to a nucleic acid-retaining separation matrix to bind the RNA
Implementation Method 6
concentrating the elution volume at least 2-fold by passing the elution volume through a semipermeable polymer membrane
Data Source
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AI summary
The invention is a novel method of isolating long nucleic acids from samples suitable for nucleic acid sequencing. The method is especially suitable for isolating low-concentration nucleic acids, e.g., viral nucleic acids, from clinical samples.