ID Sequence Pyrogram Design for Multiplex Genotyping
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Solution Overview
Problem
Current genotyping methods, such as pyrosequencing, face challenges in identifying multiple pathogens in a single sample due to overlapping peaks, making it difficult to determine the degree of infection for each pathogen, especially with repetitive sequences.
Innovation Solution
The use of an ID sequence with an ID mark, signpost, and endmark, specifically designed for pyrosequencing, allows for unique and simple pyrogram generation by separating nucleotide peaks, enabling distinct identification of genotypes and pathogens.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If pyrosequencing is used for genotyping, then DNA sequencing can be performed based on nucleotide incorporation detection, but overlapping peaks occur when multiple pathogens exist in the same sample, making genotype identification difficult
Solution Approach 1:
The patent divides the nucleotide detection process into separate channels by assigning different fluorophores (FAM, HEX, ROX) to different nucleotide types (A, C, G). This segmentation allows simultaneous detection of multiple nucleotides without peak overlap, resolving the contradiction between genotyping efficiency and measurement precision
Solution Approach 2:
The patent introduces fluorescently labeled nucleotides as intermediaries that emit distinct signals when incorporated into DNA. These fluorophore-labeled nucleotides act as mediators between the polymerase reaction and the detection system, enabling clear differentiation of multiple pathogens through wavelength-specific detection
2Reliability
If traditional genotyping methods are used, then pathogen detection can be performed, but the methods are too slow, expensive, and technically demanding for most diagnostic settings
Solution Approach 1:
The patent replaces traditional mechanical gel electrophoresis systems with an automated fluorescent detection system. The real-time monitoring of nucleotide incorporation through fluorescence emission eliminates the need for manual gel processing, significantly increasing diagnostic speed while maintaining detection accuracy
Solution Approach 2:
The patent uses pyrophosphate release detection during DNA synthesis as an accelerated reaction mechanism. The exothermic nature of nucleotide incorporation and pyrophosphate release provides a rapid signal amplification mechanism that speeds up detection without compromising accuracy
3Measurement precision
If high density microarrays are used for pathogen detection, then genetic characteristics can be detected, but the methods require cumbersome gel electrophoresis and DNA purification steps
Solution Approach 1:
The patent extracts and eliminates the cumbersome gel electrophoresis and DNA purification steps from the detection process. By using real-time fluorescent detection during PCR amplification, the method directly detects genetic characteristics without requiring separate purification and electrophoresis steps, reducing process complexity while maintaining detection precision
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 efficient genotyping of HPV, KRAS gene mutations, and respiratory viruses by producing clear pyrograms, effectively distinguishing between multiple pathogens and their respective infections.
Implementation Method 1
pyrosequencing is a method of DNA sequencing based on the 'sequencing by DNA synthesis' principle, which relies on the detection of pyrophosphate release on nucleotide incorporation
Implementation Method 2
PPi attached to the dNTPs being polymerized emit light by enzymatic reactions
Data Source
AI summary
The present invention relates to a genotyping method, and more particularly to an ID sequence, which is assigned to each genotype, and a multiplex genotyping method which uses the ID sequence. When pyrosequencing is performed using the ID sequence, a unique and simple pyrogram can be obtained for each genotype. Thus, the use of the ID sequence makes it possible to genotype viral genes, disease genes, bacterial genes and identification genes in a simple and efficient manner. In addition, a genotyping primer of the invention can be used in various genotyping methods which are performed using dispensation orders and sequencing methods.