Nucleic Acid Processing Solution for Direct PCR Template Preparation
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Solution Overview
Problem
Current nucleic acid amplification-based assays require time-consuming and toxic reagent-dependent DNA extraction methods from biological samples, which are inefficient and costly.
Innovation Solution
A method involving a nucleic acid processing solution (NAPS) with chelating, denaturing, and buffering agents is used to make nucleic acids in biological samples available for amplification without extraction, allowing direct use of the sample as a template in nucleic acid amplification reactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional DNA extraction methods using proteolytic enzymes, detergents, and organic solvents are used, then nucleic acids can be isolated from biological samples, but the process becomes time-consuming and requires toxic reagents
Solution Approach 1:
The invention extracts only the essential function of releasing nucleic acids from cells by using a simplified lytic buffer system without requiring complex extraction steps. The buffer contains salts, chelating agents, and non-ionic detergents that directly lyse cells and release nucleic acids into solution, eliminating the need for organic solvent extraction and alcohol precipitation steps.
Solution Approach 2:
The invention changes the chemical parameters of the processing solution by using a specific pH range (6.0-8.0) and ionic strength that optimizes cell lysis while maintaining nucleic acid stability. The buffer composition is adjusted to prevent protein contamination without requiring toxic reagents, enabling direct use of the processed sample in downstream applications.
2Reliability
If traditional DNA extraction methods using proteolytic enzymes and organic solvents are used, then nucleic acids can be isolated from biological samples, but toxic reagents are required
Solution Approach 1:
The invention replaces expensive and toxic reagents with a simple, disposable lytic buffer system that can be prepared from common laboratory chemicals. The buffer is designed for single-use processing where the entire buffer-sample mixture can be directly applied to spin columns or used in PCR, eliminating the need for repeated use of toxic organic solvents like phenol and chloroform.
Solution Approach 2:
The invention converts the potentially harmful effect of cell lysis into a benefit by using mild non-ionic detergents and chelating agents that simultaneously lyse cells and protect nucleic acids from degradation. The chelating agents bind metal ions that would otherwise catalyze DNA degradation, turning a potential harm into a protective mechanism.
3Reliability
If nucleic acid extraction steps are performed, then pure nucleic acids can be obtained, but sample handling requirements increase and costs increase
Solution Approach 1:
The invention merges the cell lysis step with the nucleic acid release step into a single buffer treatment. The lytic buffer simultaneously breaks down cell membranes, releases nucleic acids, and prevents protein contamination in one step, eliminating the need for separate extraction, precipitation, and washing steps required by traditional methods.
Solution Approach 2:
The lytic buffer is designed to perform multiple functions: cell lysis, nucleic acid release, protein denaturation, and inhibition of nucleases. This multi-functional buffer system replaces multiple specialized reagents and steps, allowing the same solution to be used from sample preparation through to downstream applications like PCR or sequencing.
4Reliability
If traditional nucleic acid extraction methods are used, then nucleic acids can be isolated for amplification, but the process becomes costly
Solution Approach 1:
The invention uses a cost-effective lytic buffer composed of inexpensive laboratory chemicals including salts, chelating agents, and non-ionic detergents. The buffer is designed for single-use processing where the entire buffer-sample mixture can be directly applied to spin columns or used in PCR, eliminating the need for expensive organic solvents and multiple consumption-based reagent steps.
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 reduces sample handling, time, and costs, while maintaining the integrity of nucleic acids for effective amplification, enabling quicker and more efficient diagnostic processes.
Implementation Method 1
contacting the cell sample with a nucleic acid processing solution (NAPS) having a chelating agent, a denaturing agent, and a buffering agent
Implementation Method 2
contacting the cell sample with a nucleic acid processing solution (NAPS) having a chelating agent, a denaturing agent, and a buffering agent
Data Source
AI summary
Methods are described for the processing of biological samples for direct use in nucleic acid amplification without extracting or isolating the nucleic acids from the sample. In preferred embodiments, the processed sample provides the nucleic acid template in PCR-based assays.