Double Allele Specific PCR for SNP Microarray Detection
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Solution Overview
Problem
Current methods for detecting single nucleotide polymorphisms (SNPs) are time-consuming, costly, and not suitable for large-scale genotyping due to the need for expensive instruments and specific probe design in high-throughput microarray chip technology and real-time polymerase chain reaction.
Innovation Solution
A method of double allele specific PCR for SNP microarray using SYBR® green dye as an interchelating agent, with primer sets containing four allele specific primers that recognize SNPs, allowing for multiplex SNP PCR without the need for specific nucleotides, and using universal primers with modified labels for enhanced detection specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If high-throughput microarray chip technology is used for SNP detection, then detection throughput is improved, but pre-treatment time and cost increase significantly
Solution Approach 1:
The method performs preliminary SNP-specific amplification using allele-specific primers before microarray hybridization. This pre-amplification step enriches the target sequences and reduces the complexity of subsequent processing, thereby decreasing pre-treatment time while maintaining high detection throughput
Solution Approach 2:
The detection process is segmented into distinct stages: (1) allele-specific PCR amplification to enrich target sequences, (2) product purification, and (3) microarray hybridization. This segmentation allows optimization of each step independently, reducing overall pre-treatment time while maintaining high throughput
2Measurement precision
If real-time PCR with specific probes is used for SNP detection, then detection specificity is improved, but design cost and overall detection cost increase
Solution Approach 1:
The method uses universal PCR primers flanking the SNP site that can amplify multiple SNP variants simultaneously, combined with allele-specific discrimination at the 3' end. This universal approach eliminates the need for designing separate probes for each SNP, significantly reducing design costs while maintaining high detection specificity through the allele-specific primer mechanism
Solution Approach 2:
The method changes the detection parameter from probe sequence specificity to primer 3' end nucleotide specificity. By making the 3' terminal nucleotide of primers complementary to specific alleles, the method achieves high specificity without requiring expensive probe design for each SNP variant
3Measurement precision
If traditional SNP detection methods are used, then accuracy is maintained, but detection speed and scalability for large-scale genotyping decrease
Solution Approach 1:
The method merges allele-specific PCR amplification with microarray-based detection in a coordinated workflow. The allele-specific primers ensure accurate genotyping by amplifying only the intended alleles, while the microarray platform enables parallel detection of multiple SNPs, achieving both high accuracy and high detection speed for large-scale genotyping
Solution Approach 2:
The method uses PCR amplification to create multiple copies of the target DNA sequences before detection. This amplification step increases the abundance of target molecules, enabling rapid and accurate detection by the microarray system, thereby improving both detection speed and scalability
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 method reduces the complexity and cost of primer design, enables efficient detection of multiple SNPs simultaneously, and enhances detection specificity, making it a cost-effective and rapid large-scale SNP genotyping technology.
Implementation Method 1
an interchelating agent is added into PCR, and the interchelating agent is SYBR® green dye
Data Source
AI summary
The present invention provides a method of double allele specific polymerase chain reaction (PCR) for single nucleotide polymorphism (SNP) microarray, the method provides the allele specific site as the 3′ terminal nucleotide of forward and reverse primers, it does not require a primer having specific nucleotides. The method of the present invention is easily to design the primer based on flanking region of the allele specific site, and to perform multiplex SNP PCR applying with an interchelating agent, then to detect by a SNP microarray.