Lytic Composition for Direct Nucleic Acid qPCR Analysis
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Solution Overview
Problem
Conventional DNA extraction methods for quantitative PCR are time-consuming, toxic, and not suitable for high-throughput experiments, often resulting in DNA loss and contamination, and require expensive equipment and multiple steps.
Innovation Solution
A lytic composition comprising NaOH, KOH, or Ca(OH)2 combined with PEG, which allows for rapid and simple lysis of biological samples, enabling direct nucleic acid analysis without purification, using a kit and method that includes treating samples with the composition and Turbo Buffer to release nucleic acids for qPCR.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional phenol/chloroform DNA extraction method is used, then DNA can be purified, but the method is time-consuming, complicated in extraction steps, high in toxicity, and cannot be used for high-throughput experiments
Solution Approach 1:
The invention extracts and removes the DNA purification step from the conventional workflow. Instead of using phenol/chloroform extraction followed by purification, the method directly uses crude lysate from simple lysis for qPCR, eliminating the time-consuming purification steps while maintaining sufficient DNA quality for amplification.
Solution Approach 2:
The invention merges the DNA extraction and qPCR setup steps into a single lysis step. The lysis buffer components (SDS, Proteinase K, EDTA) simultaneously lyse cells, denature proteins, and prepare DNA for direct use in qPCR without requiring separate purification and resuspension steps.
2Reliability
If ion exchange spin column method is used, then DNA can be collected, but the multi-steps are relatively complicated, centrifugal machine needs to be repeatedly used, centrifuge tubes are repeatedly replaced, DNA contamination among samples may occur, DNA is easily lost, extraction amount is small, and lab wastes which are difficult to eliminate may be produced
Solution Approach 1:
The invention removes the spin column filtration step and centrifugation steps from the workflow. DNA is collected directly in the lysis solution without requiring passage through silica membranes or repeated centrifugation, eliminating the associated equipment, consumables, and contamination risks.
Solution Approach 2:
The lysis buffer system performs multiple functions simultaneously: cell lysis, protein denaturation, and DNA stabilization. The SDS- Proteinase K-EDTA combination self-completes the extraction preparation without requiring additional purification apparatus or manual intervention for DNA isolation.
3Ease of operation
If magnet bead method is used, then DNA purification is relatively simple and convenient, but multiple steps are needed and expensive special equipment of automatic workstation is needed
Solution Approach 1:
The invention eliminates the magnet bead separation step and automatic workstation requirement. DNA is prepared directly in the lysis buffer through simple mixing and incubation, with no need for magnetic separation apparatus or automated liquid handling systems.
4Reliability
If conventional DNA extraction methods are used, then DNA can be purified, but DNA is partially lost in the experimental process and cross contamination of samples may occur due to repeated replacement of centrifuge tubes
Solution Approach 1:
The invention combines DNA lysis and qPCR template preparation into a single continuous process in the same tube. The lysate is directly transferred to the qPCR reaction mix without intermediate transfer steps, eliminating DNA loss during purification and cross-contamination from tube exchanges.
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 simplifies the nucleic acid preparation process, reduces contamination and equipment costs, and facilitates high-throughput analysis while avoiding toxic reagents, allowing for efficient and direct use of nucleic acids in qPCR without extraction, thus enhancing sample traceability and reducing waste.
Implementation Method 1
A lytic composition comprising NaOH, KOH, or Ca(OH)2 combined with PEG, which allows for rapid and simple lysis of biological samples
Data Source
AI summary
The present invention relates to the technical field of biology, and in particular relates to a lytic composition and an application thereof, a kit, a method for preparing nucleic acid by utilizing the lytic composition, and a nucleic acid analysis method. A lytic product obtained in the present invention can be directly used for the nucleic acid analysis method without purifying the nucleic acid. According to the lytic composition disclosed by the present invention, multiple biological tissues and cells are lysed so as to release the nucleic acid of the cells into a solution, the nucleic acid in a whole-cell lysing and mixing state directly serves as a template without using a conventional nucleic acid separation and purification process after termination of the reaction, and nucleic acid amplification analysis is guided. For example, nucleic acid amplification is performed by a real-time fluorescence qPCR, etc.