Soybean SSR Multiplex PCR With Fluorescent Primer Grouping
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Solution Overview
Problem
Current methods for soybean SSR marker detection are limited by low throughput and require significant reagents, hindering efficient analysis of genetic diversity and variety authentication.
Innovation Solution
A method for high-throughput multiplex PCR in soybean using a set of 28 optimized SSR primers, combined into three groups for capillary electrophoresis detection, with a standardized PCR program and fluorescent labeling for efficient fragment analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If single-locus SSR primer analysis is used, then genotype information can be obtained, but throughput is low
Solution Approach 1:
Multiple SSR primer pairs (9-10 pairs) are combined into a single multiplex PCR reaction system, allowing simultaneous amplification of multiple loci. The primers are grouped into three sets based on fluorescent labels (FAM, VIC, NED, PET), enabling parallel detection in one capillary electrophoresis run, thus dramatically increasing throughput from single-locus to multi-locus analysis.
Solution Approach 2:
The detection system is designed to handle multiple primer pairs with different fluorescent labels simultaneously. The capillary electrophoresis system with four-color fluorescence detection can universally detect all primer pairs in a single run, making the system multi-functional for high-throughput SSR marker analysis across multiple loci.
2Productivity
If multiplex PCR with multiple primer pairs is implemented, then throughput increases, but reagent consumption increases
Solution Approach 1:
Multiple primer pairs are merged into a single reaction well for multiplex PCR amplification. By combining 9-10 primer pairs in one reaction, the system reduces the number of separate reactions needed, thereby decreasing overall reagent consumption despite the increased complexity of the mixture.
Solution Approach 2:
The invention uses fluorescently labeled primers that can be detected through capillary electrophoresis, creating a copy of the genetic information in a detectable format. This allows simultaneous analysis of multiple loci without requiring proportional increases in reagent amounts for each additional locus.
3Productivity
If multiplex PCR system is established, then high throughput is achieved, but optimization difficulty increases
Solution Approach 1:
The 28 SSR primer pairs are segmented into three distinct sets based on their fluorescent labels and amplification characteristics. This segmentation strategy simplifies the optimization process by allowing independent optimization of each set before combining them into the final multiplex system, reducing the overall complexity of system establishment.
Solution Approach 2:
The invention optimizes multiple parameters including primer concentrations, annealing temperatures, and extension times to achieve successful multiplex amplification. By systematically adjusting these parameters for each primer set and then for the combined system, the invention overcomes the optimization challenges inherent in multiplex PCR.
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 significantly reduces experimental costs and improves detection efficiency by allowing simultaneous amplification and analysis of multiple SSR markers in a single well, enhancing genetic diversity analysis and variety authentication.
Implementation Method 1
conducting PCR amplification; a procedure of the PCR amplification includes: initial denaturation at 95° C. for 3 min; 32 cycles for a process of denaturation at 95° C. for 30 s and then annealing at 60° C. for 4 min; and extension at 72° C. for 5 min
Implementation Method 2
initial denaturation at 95° C. for 3 min; 32 cycles for a process of denaturation at 95° C. for 30 s
Implementation Method 3
annealing at 60° C. for 4 min
Implementation Method 4
extension at 72° C. for 5 min; 12.5 μL of Premix Ex Taq Hot Start enzyme
Implementation Method 5
subjecting an obtained amplified product to capillary electrophoresis, and then detecting a length of an obtained fragment
Data Source
AI summary
A method for high-throughput multiplex PCR in soybean using a simple sequence repeat (SSR) marker is provided, relating to the technical field of molecular biology. 28 pairs of SSR primers that can be used for multiplex PCR amplification and capillary electrophoresis detection are screened out of 38 pairs of SSR primers, in which 28 SSR markers can be divided into 3 groups. Compared with traditional SSR marker detection, 9-10 pairs of primers in the developed SSR markers can complete PCR amplification and capillary electrophoresis in one well, greatly reducing experimental cost and improving detection efficiency. Different primers are labeled with different fluorescent groups, and a length of an amplified fragment at each SSR site for each soybean material is obtained by the capillary electrophoresis.