DNA Tag Design for Specific Primer Binding in Microorganism Monitoring
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Solution Overview
Problem
Current methods for monitoring microorganism spread in bioremediation face challenges due to non-specific primer reactions with environmental DNA sequences, making it difficult to accurately assess microorganism numbers and cleanup progress, especially in environments with diverse organisms, and are restricted by regulations regarding recombinant organisms.
Innovation Solution
A method is developed to determine a DNA tag with low frequency in natural environments and introduce it into microorganisms, using a program that selects a DNA sequence and primer with specific characteristics to enhance primer specificity in PCR, allowing for accurate monitoring without categorizing the modified microorganism as a recombinant organism under the Cartagena Law.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a DNA sequence is used as a primer for quantitative PCR to monitor microorganism spread, then the microorganism population can be measured, but the primer may react with non-specific DNA sequences in environmental DNA, reducing measurement accuracy
Solution Approach 1:
The invention changes the nucleotide sequence parameters of the primer by introducing silent mutations (synonymous substitutions) that do not alter the encoded amino acid sequence. This modifies the primer's binding characteristics to environmental DNA while maintaining the microorganism's genetic function, thereby improving specificity and measurement accuracy
Solution Approach 2:
The invention performs preliminary computational analysis to identify and select optimal silent mutation sites in the DNA sequence before implementing the actual mutation. This preliminary screening ensures that the selected mutation sites will provide high primer specificity while maintaining protein function, resolving the contradiction between measurement accuracy and primer reliability
2Measurement precision
If an artificial DNA sequence is introduced into a microorganism to improve primer specificity, then monitoring accuracy improves, but the microorganism may be classified as a recombinant organism under the Cartagena Law, creating regulatory restrictions
Solution Approach 1:
The invention uses silent mutations that change the nucleotide sequence parameters without altering the amino acid sequence or protein function. Since the protein-coding capability remains unchanged, the modified microorganism does not meet the definition of a recombinant organism under the Cartagena Law, thereby maintaining regulatory compliance flexibility while improving monitoring accuracy
Solution Approach 2:
The invention creates a copy of the original DNA sequence with synonymous substitutions that preserve the original protein-coding information. This copying approach with silent mutations allows the microorganism to maintain its natural genetic characteristics while providing a unique marker for accurate monitoring, avoiding recombinant organism classification
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 enables precise monitoring of microorganism dispersion and cleanup progress while avoiding regulatory issues, ensuring high primer specificity and compliance with environmental regulations.
Implementation Method 1
a step S4 of obtaining one or more third coding sequences produced by subjecting the corresponding second coding sequences obtained in the step S3 to a silent mutation
Implementation Method 2
performing a quantitative polymerase chain reaction (PCR) using, as a primer, a DNA sequence specific to the microorganism
Data Source
AI summary
Provided is a method of determining a DNA tag which is a base sequence to be introduced into a genomic DNA sequence of an organism and an introduction site of the DNA tag into the genomic DNA sequence. The method includes: a step (S1) of obtaining a protein coding sequence of the genomic DNA sequence; a step (S2) of determining a region to be treated in the protein coding sequence; a step (S3) of fragmenting the coding sequence in the region to be treated; a step (S4) of subjecting the fragmented coding sequences to a silent mutation; a step (S5) of determining sequences suitable as primers from polynucleotides including the fragmented coding sequences or complementary base sequences thereof; a step (S6) of performing a homology search for the coding sequences thus determined; and a step (S8) of determining the determined coding sequence corresponding to a minimum value of an NMS as the DNA tag, and determining the site of the fragmented coding sequence corresponding to the determined coding sequence in the protein coding sequence as the introduction site.