PCR Primer Set for Whole Genome Sequencing
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Solution Overview
Problem
Current whole genome sequencing methods for plant breeding are hindered by high development costs, limited detection of new SNP sites, dependency on instrument-based processes, and inefficiencies in library establishment, particularly requiring high-quality DNA and uniform sequencing quantities.
Innovation Solution
A sequencing primer set comprising a universal upstream primer, a universal downstream primer, and a downstream primer in rich of promoter, optimized for PCR-based whole genome sequencing, which allows for the detection and enrichment of SNP sites without the need for pre-existing SNP markers or high-quality DNA, enabling simultaneous amplification and sequencing with reduced costs and simplified library construction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If chip-based genotyping methods are used, then detection precision of SNP sites is improved, but development cost and instrument dependency increase
Solution Approach 1:
The patent replaces expensive, reusable chip-based systems with inexpensive, disposable PCR primers. The primers are simple oligonucleotides that can be synthesized cheaply and do not require complex instrumentation, thereby reducing both cost and instrument dependency while maintaining SNP detection capability
Solution Approach 2:
The patent extracts the essential function of SNP detection from the complex chip-based system and implements it through simplified PCR amplification using specific primers. This separates the detection function from the complex instrumentation, allowing it to be performed with basic PCR equipment
2Quantity of substance
If restriction enzyme digestion is used for simplified sequencing, then sequencing cost is reduced, but library establishment complexity and time increase
Solution Approach 1:
The patent extracts the essential function of genome reduction from complex restriction enzyme digestion and library construction processes, and implements it through direct PCR amplification using genome-wide distributed primers. This eliminates the need for enzyme digestion, gel purification, and complex library preparation steps
Solution Approach 2:
The PCR primers are designed to directly amplify genomic DNA without requiring prior processing steps. The primers themselves perform the function of both reducing genome complexity and enabling sequencing, eliminating the need for separate library establishment procedures
3Measurement precision
If high-quality DNA is used for sequencing, then sequencing precision is improved, but sample preparation time and cost increase
Solution Approach 1:
The patent changes the parameter of DNA quality requirements by designing primers that can amplify from lower-quality genomic DNA. The primers are engineered with features that allow them to bind effectively even when DNA is degraded or of variable quality, thereby reducing the need for extensive DNA purification while maintaining amplification precision
Solution Approach 2:
The patent uses simple, inexpensive PCR primers that can tolerate variations in DNA quality. These primers do not require the high-quality DNA input that traditional sequencing methods demand, allowing direct amplification from crude genomic extracts without time-consuming purification steps
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 rapid, cost-effective detection and screening of whole genomes, reducing sequencing time and improving library construction efficiency, with high coverage and enrichment of gene regions, making it suitable for diverse samples and reducing the need for instrument-dependent chip-based methods.
Implementation Method 1
conducting the first PCR amplification on the DNA of the sample to be tested by using the universal upstream primer and the universal downstream primer to obtain the first amplification product
Implementation Method 2
conducting the second PCR amplification on the DNA of the sample to be tested by using the universal upstream primer and the downstream primer in rich of promoter to obtain the second amplification product
Data Source
AI summary
A sequencing primer set comprising a universal upstream primer, a universal downstream primer and a downstream primer in rich of promoter; wherein the universal upstream primer has a sequence of: 5′-TBarcodeCAAAXXXXNNN-3′; the universal downstream primer has a sequence of: 5′-GACTGCGTACGZZZZNNN-3′(SEQ ID No. 9); the downstream primer in rich of promoter has a sequence of: 5′-GACTGCGTACYYNCTATA-3′(SEQ ID No. 7).