Dried Hot-Start PCR Composition Stabilized with Polyols and Pyrophosphatase
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Solution Overview
Problem
Conventional hot-start PCR methods face challenges with non-specific amplification, contamination, and limited applicability due to instability and storage issues, particularly with pyrophosphatase (PPase) degrading at room temperature, leading to decreased hot-start PCR effectiveness and increased complexity in handling.
Innovation Solution
A dried composition for hot-start PCR is developed by combining inorganic pyrophosphate (PPi) and PPase with reaction components like DNA polymerase, dNTPs, and a reaction buffer, which is then stabilized with polyols and non-reactive dyes, allowing for long-term storage and improved reproducibility.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If PPase is added to PCR composition to prevent non-specific amplification, then PCR specificity is improved, but PPase degrades at room temperature leading to limited storagability
Solution Approach 1:
The patent changes the physical state of the PCR composition from liquid to dried form. This parameter change stabilizes PPase at room temperature, allowing long-term storage without degradation while maintaining its ability to prevent non-specific amplification when reconstituted
Solution Approach 2:
The patent performs drying of the PCR composition containing PPase in advance, before use. This preliminary action stabilizes the enzyme for storage, and the composition is reconstituted with water immediately before PCR to restore PPase activity for preventing non-specific amplification
2Reliability
If paraffin wax is used as physical barrier for hot-start PCR, then high stringency priming is facilitated, but the method is troublesome and time-consuming
Solution Approach 1:
The patent removes the paraffin wax barrier component from the hot-start PCR system. Instead of using physical barriers, the invention relies on the chemical mechanism of PPase inhibiting non-specific priming at low temperatures, which is then activated by heating, thereby simplifying the procedure while maintaining specificity
Solution Approach 2:
The patent replaces the mechanical/physical barrier system (paraffin wax that must be melted and removed) with a chemical/enzymatic system (PPase that is activated by temperature). This substitution eliminates the need for physical barrier manipulation, making the process simpler and faster
3Duration of action of stationary object
If mineral oil is used as evaporation barrier in hot-start PCR, then long-term storagability is secured, but oil contaminates PCR samples making quantitative analysis difficult
Solution Approach 1:
The patent removes mineral oil from the PCR composition entirely. Instead of using oil as an evaporation barrier, the invention uses a dried composition that can be stored without oil, and reconstituted with water before use, thereby eliminating oil contamination while maintaining storagability
Solution Approach 2:
The patent employs a dried composition that is stable during storage but requires reconstitution immediately before use. This approach treats the active composition as a short-living component that is prepared fresh for each experiment, eliminating the need for long-term stable formulations with stabilizing additives like mineral oil
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 stabilized dried composition enhances PCR specificity, reduces non-specific amplification, and facilitates convenient use by eliminating the need for pre-treatment at high temperatures, while maintaining reactivity and stability over time, making it suitable for diverse gene diagnosis and tests.
Implementation Method 1
adding a required amount of polyols, inorganic pyrophosphate (referred as 'PPi' hereinafter), and pyrophosphatase (referred as 'PPase' hereinafter) to the conventional PCR composition
Implementation Method 2
a dried composition for hot-start PCR which is stabilized and has a long-term storagability (shelf-life) by adding a required amount of polyols
Implementation Method 3
Hot-start PCR is a kind of PCR to obtain a more pure PCR product, which allows high temperature mixing of each reactant, so that PCR specificity can be increased by preventing low stringency priming frequently occurring at room temperature
Data Source
AI summary
The present invention relates to a dried composition for hot-start PCR, more precisely a dried composition for hot-start PCR with improved stability and long-term storagability which is characteristically prepared by the steps of preparing a reaction mixture by mixing an aqueous solution containing reaction buffer, MgCl2, 4 types of dNTPs, DNA polymerase with pyrophosphate and pyrophosphatase in a reaction tube; and drying the reaction mixture prepared above, a preparation method of the same and a method for amplifying nucleic acid using the same. The dried composition for hot-start PCR is added with pyrophosphate and pyrophosphatase together before drying, so that it can have improved stability and long-term storagability as well as convenience in use, compared with the conventional compositions for hot-start PCR. Therefore, this composition can be effectively used for hot-start PCR, multiplex PCR or real-time quantitative PCR.