Lyophilized PCR Reagent Kit with Cyclodextrin for Direct Nucleic Acid Amplification
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Solution Overview
Problem
Current methods for DNA amplification through PCR are complex, costly, and prone to contamination, with existing lyophilization and freeze-drying techniques facing challenges in achieving homogeneous blends and reproducibility, particularly for labile reagents used in molecular biology.
Innovation Solution
A lyophilized PCR reagent composition comprising a sequestering agent like cyclodextrin, polymerase, and dNTPs, along with primers and excipients, which can be easily reconstituted at room temperature, reducing the number of steps and minimizing contamination risks.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional DNA purification methods (column-based extraction) are used, then nucleic acid can be purified, but the process becomes complex, time-consuming, and prone to contamination
Solution Approach 1:
The invention extracts and removes the purification step entirely from the PCR process. By formulating a direct lysis/PCR method where cells are lysed directly in the PCR tube and the resulting nucleic acid is used immediately without column-based purification, the complex purification process is eliminated. This reduces contamination risk and simplifies the overall workflow while maintaining amplification effectiveness.
Solution Approach 2:
The invention merges the cell lysis step with the PCR amplification step into a single integrated process. The lysis reagents are combined with PCR reagents in the same tube, allowing direct amplification of nucleic acid released from lysed cells. This consolidation eliminates the need for separate purification steps and reduces the number of handling operations that could introduce contamination.
2Reliability
If multiple purification steps are performed, then nucleic acid purity is improved, but operator time and complexity increase
Solution Approach 1:
The invention performs preliminary cell lysis and nucleic acid release directly in the PCR tube before amplification begins. By pre-lysing the cells in the presence of PCR reagents and using the resulting mixture directly for amplification without intermediate purification, the method eliminates time-consuming manual steps while ensuring the nucleic acid is in a suitable state for PCR.
3Stability of the object's composition
If lyophilized PCR reagents are prepared, then storage and shipping stability is improved, but achieving homogeneous blends and reproducibility becomes challenging
Solution Approach 1:
The invention changes the physical state of the PCR reagents from liquid to lyophilized (freeze-dried) form. This parameter change enables stable storage and shipping at room temperature without refrigeration. The lyophilization process removes water from the reagent mixture, creating a stable powder that can be reconstituted by adding water or buffer immediately before use, thereby achieving both stability and operational convenience.
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 PCR process, reduces operator time, and enhances reproducibility by providing a stable, easy-to-use reagent mixture that can be stored and shipped at room temperature, minimizing pipetting errors and contamination.
Implementation Method 1
the addition of cyclodextrin to neutralize the detergent removes the need for the separation step
Implementation Method 2
The invention provides methods and kits which can be used to amplify nucleic acids by lyophilizing or freeze-drying nucleic acid amplification reagents
Data Source
AI summary
The present invention relates to compositions, methods and kits which can be used to amplify nucleic acids with the advantage of decreasing user time and possible contamination. The dried reagent composition of the invention can be used for easy processing and amplification of nucleic acid samples.