Nucleotide Sequence Profiling for Microorganism Identification
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Solution Overview
Problem
Conventional methods for microorganism identification, such as biochemical and phenotypic techniques, are slow and lack discriminatory power, while existing genotypic techniques like DNA sequencing are costly and require full sequencing, which is not always necessary for identification.
Innovation Solution
A method involving nucleotide sequence profiling that extracts a nucleotide sequence template, performs amplification via PCR, conducts chemical cleavage on the amplified template to generate fragments, separates and detects these fragments by size, and compares the resulting profile to a database for identification, allowing for efficient identification without full DNA sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional biochemical and phenotypic techniques are used for microorganism identification, then the methods are effective for identification, but the process is slow and takes more than 24 hours to generate accurate identification
Solution Approach 1:
The invention extracts only the essential discriminatory information from the genome by performing chemical cleavage at specific nucleotide positions (A, G, C, or T) to generate a reduced set of fragments that contain sufficient taxonomic information without requiring complete sequencing of the entire genome region
Solution Approach 2:
Instead of performing complete DNA sequencing, the method performs partial sequencing by chemically cleaving the amplified DNA at less than four different nucleotide positions, generating a profile that is sufficient for accurate microorganism identification while significantly reducing the time and resources required
2Measurement precision
If genotypic techniques with full DNA sequencing are used for microorganism identification, then high-degree of accuracy is provided, but the cost is high and full sequencing is not always necessary
Solution Approach 1:
The invention extracts only the essential discriminatory information from the genome by performing chemical cleavage at specific nucleotide positions (A, G, C, or T) to generate a reduced set of fragments that contain sufficient taxonomic information without requiring complete sequencing of the entire genome region
Solution Approach 2:
The method uses chemical cleavage reactions with readily available chemical agents (such as formic acid for A-cleavage, hydroxylamine for G-cleavage, etc.) that are inexpensive and can be performed in a single reaction tube, eliminating the need for expensive fluorescently-labeled terminators and multiple sequencing reactions
3Loss of information
If chemical cleavage reactions are performed on amplified DNA template, then nucleotide sequence information is obtained for identification, but four different reactions are traditionally required which increases complexity
Solution Approach 1:
The invention merges multiple chemical cleavage reactions into a single reaction tube by performing sequential or simultaneous cleavage at different nucleotide positions using different chemical agents, allowing all four nucleotide positions (A, G, C, T) to be interrogated in one experiment rather than requiring four separate reactions
Solution Approach 2:
The method uses a universal approach where the same basic chemical cleavage methodology can be applied to any nucleotide position by selecting the appropriate chemical agent, making the system versatile and eliminating the need for position-specific reaction conditions or specialized reagents
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
Enables rapid and accurate identification of microorganisms and genetic mutations, reducing costs and time, while providing sufficient discriminatory power to differentiate between species and strains, thus aiding in targeted therapy selection.
Implementation Method 1
The PCR amplifies a section of genome that is characterized through the traditional DNA sequencing reaction
Implementation Method 2
The PCR product is then submitted to the Sanger sequencing reaction (i.e., enzymatic chain elongation/termination through the incorporation of di-deoxyribonucleotide triphosphate [ddNTP] incorporation)
Implementation Method 3
The products are then separated by electrophoresis and differentiated with laser-induced fluorescence detection
Implementation Method 4
differentiated with laser-induced fluorescence detection
Implementation Method 5
Maxam and Gilbert have developed a sequencing method based on chemical reactions. This reaction procedure determines the nucleotide sequence of a terminally labeled DNA molecule by random breaking at adenine (A), guanine (G), cytosine (C), or thymine (T) positions using specific chemical agents
Data Source
AI summary
The invention includes a method of sample profiling for identification. The method includes the steps of performing less than four nucleotide-specific chemical cleavage reactions to obtain nucleotide sequence fragments, performing size separation on the fragments, detecting the fragments' separation, generating a profile based on the detection, and comparing the profile to a data base to identify the sample.