Exonuclease Treatment for Nucleic Acid Amplification Contamination
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Solution Overview
Problem
Nucleic acid amplification processes, such as PCR and LCR, face contamination issues due to carryover of amplification products, leading to false positives and increased costs, as these products can serve as substrates for subsequent amplifications, contaminating laboratory environments.
Innovation Solution
Incorporating exo-sample nucleotides into amplification products, which can be selectively destroyed, rendering them inactive as templates for further amplification, thereby distinguishing them from natural DNA and preventing contamination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If amplification products are used as substrates for subsequent amplifications, then productivity is improved, but contamination occurs leading to false positives
Solution Approach 1:
The patent applies preliminary action by treating amplification products with exonuclease before subsequent amplifications to remove contaminating nucleic acid sequences. This pre-treatment step eliminates contamination risks while maintaining productivity, as the exonuclease digestion occurs beforehand and does not interfere with the amplification throughput.
Solution Approach 2:
The patent introduces exonuclease as an intermediary substance that selectively degrades contaminating amplification products without affecting the amplification process itself. This mediator enables the system to maintain both high productivity and result accuracy by facilitating the removal of contaminants through enzymatic digestion.
2Reliability
If extreme care is taken to avoid carryover contamination, then reliability is improved, but device complexity and operational difficulty increase
Solution Approach 1:
The patent applies self-service by using exonuclease to automatically degrade contaminating amplification products in the reaction mixture itself, eliminating the need for complex external decontamination procedures. This self-contained approach simplifies the overall process while maintaining high reliability in contamination prevention.
Solution Approach 2:
The patent replaces mechanical or manual decontamination methods with enzymatic digestion using exonuclease. This substitution simplifies the process by using a biochemical mechanism instead of complex physical or operational procedures, reducing device complexity while improving reliability.
3Reliability
If amplification products are eliminated to prevent contamination, then reliability is improved, but loss of substance occurs
Solution Approach 1:
The patent applies local quality by using exonuclease to selectively degrade only the contaminating amplification products while leaving the target nucleic acid sequences intact. This localized action ensures that only the harmful substances are removed, minimizing overall substance loss while maintaining high reliability in contamination elimination.
Solution Approach 2:
The patent changes the chemical parameters of the reaction mixture by introducing exonuclease, which selectively modifies the contaminating nucleic acid sequences through enzymatic digestion. This parameter change enables selective removal of contaminants without affecting the target substances, reducing overall loss while improving reliability.
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 method effectively eliminates contamination from previous amplification products, ensuring accurate and reliable subsequent amplification reactions without affecting the amplification of authentic nucleic acids, thus reducing the risk of false positives and operational costs.
Implementation Method 1
treating the strands with exonuclease, thereby rendering the amplified nucleic acid substantially unamplifiable
Data Source
AI summary
This invention relates to a method of incorporating an exo-sample nucleotide into the amplified product strands resulting from a nucleic acid amplification process. Once the product strands have been obtained and analyzed (e.g., by hybridization, Southern blot, etc.), the exo-sample strands can be selectively destroyed by acting on the incorporated exo-sample nucleotide.Two embodiments are presented. In a first embodiment, the exo-sample nucleotide is incorporated by carrying out the amplification to reaction in the presence of an excess of exo-sample nucleotide tri-phosphate.In a second embodiment, the exo-sample nucleotide is incorporated by carrying out the amplification reaction in the presence of an oligonucleotide which has, as part of its sequence, one or more exo-sample nucleotides.