Lyophilized Nucleic Acid Polymerase Stabilization with Cellobiose
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Solution Overview
Problem
Current methods for stabilizing nucleic acid polymerases, particularly during lyophilization and storage, result in significant enzyme inactivation, limiting their use in PCR reactions and requiring stringent temperature control, which complicates storage and transportation.
Innovation Solution
A lyophilized composition containing cellobiose as a stabilizer, which maintains nucleic acid polymerase activity at temperatures up to 55°C, allowing for the creation of a universal master mix suitable for various PCR applications, including PCR, Real-Time PCR, and other molecular biology techniques.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If polymerases are stored in aqueous solutions at room temperature or +4°C, then ease of operation is improved, but enzyme activity is rapidly lost
Solution Approach 1:
The patent applies parameter changes by transitioning the storage state of polymerases from aqueous liquid to lyophilized (dry) form. This fundamental state change allows the enzyme to maintain activity at higher temperatures and for extended periods without refrigeration, resolving the contradiction between ease of storage and enzyme activity preservation
Solution Approach 2:
The patent uses composite materials by combining polymerases with protective matrices and stabilizing agents in the lyophilized formulation. This composite structure protects the enzyme from denaturation and maintains catalytic activity during storage at room temperature, simultaneously achieving ease of operation and reliability
2Temperature
If polymerases are lyophilized without adequate stabilizers, then storage temperature requirements are reduced, but significant enzyme inactivation occurs
Solution Approach 1:
The patent introduces intermediary substances (protective matrices and stabilizers) that mediate between the polymerase enzyme and the harsh lyophilization process. These intermediaries protect the enzyme structure during drying and storage, enabling low-temperature storage while maintaining enzyme activity, thus resolving the contradiction between temperature reduction and reliability
3Reliability
If stringent temperature control is implemented for polymerase storage, then enzyme activity is maintained, but device complexity and transportation costs increase
Solution Approach 1:
The patent extracts the temperature control requirement from the storage system by developing a lyophilized formulation that is inherently stable at room temperature. This removes the need for complex refrigeration equipment and temperature monitoring systems, reducing device complexity while maintaining enzyme activity through the robust lyophilized state
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 use of cellobiose in the lyophilized composition significantly enhances the stability and activity of nucleic acid polymerases, enabling reliable PCR reactions at elevated temperatures and simplifying storage and transportation, while maintaining enzyme activity for extended periods.
Implementation Method 1
The present invention relates to an exsiccated or lyophilized composition suitable for being diluted with an appropriate solvent, comprising a nucleic acid polymerization enzyme stabilized to withstand lyophilisation and storage at a temperature of up to 55° C.
Data Source
AI summary
The present invention relates to a exsiccated or lyophilized composition comprising: a nucleic acid polymerization enzyme and cellobiose, in which the enzyme is stable for a period of time, even at a temperature of up to 55° C. The composition of the invention can also comprise further reagents, such as salts, primers specific for a template DNA present in a sample, probes, etc. The invention relates to a method for preparing an exsiccated or lyophilized composition comprising a nucleic acid polymerization enzyme and cellobiose, possibly in containers, in which the enzyme is lyophilized and ready for use in molecular biology applications upon addition of the sample.