PCR Master Mix Stability and Inhibitor Tolerance
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Solution Overview
Problem
Current PCR master mixes face challenges in providing reliable and reproducible results, especially when dealing with nucleic acid samples containing inhibitors that hinder amplification, and they often require immediate use after assembly due to instability at room temperature.
Innovation Solution
The development of compositions and kits that include a thermostable DNA polymerase and a PCR inhibitor blocking agent, such as albumin and gelatin, which enhance the tolerance of PCR reactions to inhibitors and maintain stability for up to 72 hours at room temperature, allowing for sensitive and rapid nucleic acid amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If PCR master mixes are formulated to be stable at room temperature, then storage and handling convenience is improved, but amplification sensitivity and speed may deteriorate
Solution Approach 1:
The patent modifies the chemical composition parameters of the PCR master mix by incorporating specific stabilizing agents and optimizing buffer compositions. This allows the reaction mixture to maintain stability at room temperature while preserving the enzymatic activity of DNA polymerase for rapid amplification when heated to PCR temperatures.
Solution Approach 2:
The invention creates a composite PCR master mix formulation that combines multiple components including stabilizing agents, buffers, dNTPs, and DNA polymerase in specific ratios. This composite formulation achieves both room temperature stability and high amplification efficiency by synergistic interaction of its components.
2Productivity
If PCR master mixes are designed for rapid amplification, then productivity is improved, but stability during storage and handling deteriorates
Solution Approach 1:
The patent optimizes reaction parameters including temperature profiles, cycle times, and component concentrations to enable rapid amplification. Simultaneously, it adjusts stabilizing component concentrations to maintain formulation integrity during storage, achieving both speed and stability through coordinated parameter optimization.
3Adaptability or versatility
If PCR reactions are performed with samples containing inhibitors, then versatility in sample handling is improved, but amplification reliability deteriorates
Solution Approach 1:
The patent introduces inhibitor-blocking agents as intermediary substances in the PCR master mix. These agents specifically bind to or neutralize inhibitory compounds commonly found in clinical and environmental samples, preventing them from interfering with DNA polymerase activity while allowing the amplification reaction to proceed reliably.
Solution Approach 2:
The invention incorporates readily available, cost-effective inhibitor-blocking agents that can be included in the master mix formulation. These agents effectively counteract sample inhibitors without requiring complex sample purification steps, maintaining reliability while enhancing versatility in handling diverse sample types.
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
These compositions provide superior sensitivity, accuracy, and stability, enabling reliable PCR results even in the presence of inhibitors and allowing for extended storage and reduced PCR run times, making them suitable for high-throughput applications.
Implementation Method 1
enzymatic synthesis of specific DNA sequences using two oligonucleotide primers that hybridize to opposite strands and flank the region of interest in the target DNA. A repetitive series of reaction steps involving template denaturation, primer annealing, and the extension of the annealed primers by DNA polymerase results in the exponential accumulation of a specific fragment
Implementation Method 2
The PCR inhibitor blocking agent relieves inhibition of PCR caused by a variety of compounds often found in samples containing nucleic acids that are analyzed by PCR
Implementation Method 3
The PCR master mix should also be stable on the bench top at ambient or room temperature so that the PCR reactions need not be amplified immediately after assembly. The composition also provides assembled PCR reactions that may be stable for up to 72 hours or more at room temperature
Data Source
AI summary
A composition comprising a thermostable DNA polymerase; and a PCR inhibitor blocking agent, wherein the PCR inhibitor blocking agent is present in an amount effective to enhance tolerance of an assembled PCR to a PCR inhibitor.