SNP Detection in SMA Gene via Primer Extension and Mass Spectrometry
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Solution Overview
Problem
Current methods for detecting genetic defects in the SMN1 gene, such as those causing spinal muscular atrophy, suffer from low sensitivity, high cost, and poor efficiency, particularly in identifying homozygous deletions.
Innovation Solution
A method involving PCR, dephosphorylation, extension reactions, and molecular weight measurement using mass spectrometry or fluorescent electrophoresis to detect single nucleotide polymorphisms (SNPs) in the SMN1 gene, allowing for simultaneous detection of multiple SNPs and accurate identification of homozygous deletions in small specimen amounts.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If quantitative polymerase chain reaction or gene sequencing is used to detect SMN1 gene defects, then detection can be performed, but sensitivity is low, cost is high, and efficiency is poor
Solution Approach 1:
The invention segments the detection process into two distinct stages: first amplifying the target DNA sequence using PCR, then performing extension reactions at multiple SNP sites with specifically designed extension primers. This segmentation allows each stage to be optimized independently, improving both sensitivity and efficiency compared to traditional single-step methods
Solution Approach 2:
The invention introduces extension primers as intermediary elements that bind to specific SNP sites on the amplified DNA. These extension primers serve as mediators between the PCR amplification step and the final detection step, enabling precise identification of homozygous deletions through extension reactions that add detectable markers
2Reliability
If traditional detection methods are used, then detection can be performed, but the cost is high
Solution Approach 1:
The invention uses disposable extension primers with unique sequences for each SNP site that are synthesized at low cost. These primers are designed to be used in a single reaction cycle and then discarded, eliminating the need for expensive reusable equipment or complex reagent systems while maintaining high detection accuracy
Solution Approach 2:
The invention changes the detection parameter from measuring overall gene quantity (as in quantitative PCR) to detecting specific extension products at SNP sites. This parameter change allows the use of simpler, cheaper detection methods such as gel electrophoresis or capillary electrophoresis instead of expensive real-time PCR instrumentation
3Quantity of substance
If detection methods are used with limited specimen amounts, then detection can be attempted, but sensitivity is insufficient for accurate detection
Solution Approach 1:
The invention performs preliminary PCR amplification of the target DNA sequence before conducting extension reactions at SNP sites. This preliminary action increases the quantity of target DNA from limited specimens, ensuring sufficient material for subsequent detection steps while maintaining the integrity and accuracy of the original sample
Solution Approach 2:
The invention implements a nested detection strategy where extension primers are designed to bind within the PCR-amplified region. This nesting approach allows the extension reactions to occur on already-amplified DNA, effectively multiplying the signal from limited specimens and improving detection accuracy without requiring additional sample material
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 significantly reduces analysis costs, is rapid and applicable to various specimen types, and efficiently detects deletions correlated with spinal muscular atrophy, facilitating quicker genotype identification and improved study of the condition.
Implementation Method 1
performing a polymerase chain reaction (PCR) on a specimen, wherein a nucleic acid fragment containing a SNP site in the specimen is amplified by using a pair of amplification primers
Implementation Method 2
performing a dephosphorylation reaction on the amplified nucleic acid fragment to remove a phosphate at a 5′ end of the nucleic acid fragment in the PCR
Implementation Method 3
performing an extension reaction on the amplified nucleic acid fragment, wherein the SNP site is identified by using an extension primer, a 3′-end of the extension primer is extended by a single nucleotide which is complementary to a base of the SNP site
Implementation Method 4
measuring a molecular weight of the extended extension primer, and determining a type of a base of the single nucleotide based upon the molecular weight
Implementation Method 5
measuring a molecular weight of the extended extension primer, and determining a type of a base of the single nucleotide based upon the molecular weight, thereby determining whether deletion occurs to the SNP site
Data Source
AI summary
A method for detecting a SNP site on a SMA gene is disclosed, and includes steps of: (S10) performing a PCR for amplifying a nucleic a nucleic acid fragment containing a SNP site; (S20) performing a dephosphorylation reaction on the nucleic acid fragment; (S30) performing an extension reaction on the nucleic acid fragment, wherein the SNP site is identified by using an extension primer, a 3′-end of the extension primer is extended by a single nucleotide which is complementary to a base of the SNP site, and thus an extended extension primer is obtained; (S40) performing a purification reaction; and (S50) measuring a molecular weight of the extended extension primer, and determining a type of a base of the single nucleotide based upon the molecular weight, thereby determining whether deletion occurs to the SNP site.