Multiplex PCR Kit for Pig SNP Detection
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Solution Overview
Problem
Current SNP detection technologies based on PCR-RFLP can only detect one SNP at a time, making it inefficient for detecting multiple sites related to pig growth traits, which require sequential PCR amplification and electrophoresis for each site.
Innovation Solution
A reagent and kit using specific primer pairs for WIP1, TRIM55, and GAS7 genes, combined with restriction endonucleases like ScaI-HF, BstUI, and HindIII, allow for simultaneous PCR amplification and digestion to identify SNPs in multiple sites without sequencing, enabling differentiation between wild type, homozygous mutant, and heterozygous mutant types.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If PCR-RFLP is used to detect SNP sites, then detection specificity is improved, but detection efficiency deteriorates when multiple sites need to be tested
Solution Approach 1:
The patent combines multiple primer pairs targeting different SNP sites into a single PCR reaction system. Multiple primers are mixed together in one reaction tube with template DNA and polymerase, allowing simultaneous amplification of multiple gene regions. This merging approach maintains the specificity of individual primer-site interactions while achieving parallel detection of multiple SNPs, thus resolving the contradiction between detection precision and efficiency.
Solution Approach 2:
The invention creates a universal detection system where a single PCR reaction mixture can detect multiple different SNP sites associated with pig growth traits. The system is designed to handle various gene targets (growth hormone, insulin-like growth factor, etc.) using the same basic protocol and reagent composition, making the method multi-functional and highly efficient for comprehensive genetic analysis.
2Measurement precision
If multiple SNP sites are detected sequentially using traditional PCR-RFLP, then detection accuracy is maintained, but detection time increases
Solution Approach 1:
The patent implements continuous useful action by performing multiple SNP amplifications simultaneously in a single PCR reaction that runs continuously without interruption. Unlike sequential methods where reactions are performed one after another, the multi-primer system allows all target regions to be amplified in parallel during one continuous thermal cycling process, significantly reducing total detection time while maintaining accuracy through the same rigorous amplification and analysis protocols.
3Reliability
If multiple PCR amplifications are performed for different sites, then detection reliability is improved, but operation complexity increases
Solution Approach 1:
The patent merges multiple separate amplification operations into a single combined PCR reaction. By mixing multiple primer pairs in one reaction tube, the method reduces the number of separate操作步骤 from multiple sequential reactions to a single operation, thereby simplifying the workflow while maintaining the reliability that comes from thorough amplification and analysis of each target site within the unified reaction system.
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
The method provides simple, intuitive identification of pig economic trait-associated mutation sites with high specificity and sensitivity, allowing for simultaneous detection of multiple SNPs in a single assay.
Implementation Method 1
the resulting amplified product is digested with a restriction endonuclease
Implementation Method 2
amplifying a target gene fragment of a pig by PCR
Implementation Method 3
The RFLP can be adopted to detect mutations in DNA sequences. When the DNA sequences from different individuals are digested with the same enzyme and subjected to gel electrophoresis
Data Source
AI summary
A reagent, a kit, and a method for detecting a pig economic trait-associated mutation site are provided, belonging to the technical field of molecular markers. The reagent for detecting a pig economic trait-associated mutation site is provided, and the reagent and an endonuclease are prepared into the kit for detecting a pig economic trait-associated mutation site. The method for detecting a pig economic trait-associated mutation site is also provided. After PCR amplification, gene fragments of WIP1, TRIM55, and GAS7 of a pig show site differences between different individuals. Restriction endonucleases are used for digestion, and PCR or multiplex PCR analysis is conducted to identify single nucleotide polymorphisms (SNPs) of the pig, such that the wild type, the homozygous mutant type, and the heterozygous mutant type that are difficult to differentiate in morphology are differentiated from the molecular biology level.