The same drug has a significant effect on some people, but a weak effect on others. One of the most influential factors of this phenomenon is the variability in the human genome; often referred to as single nucleotide polymorphisms (SNP).

What is an SNP?

A single nucleotide polymorphism (SNP) refers to the polymorphism of DNA sequence caused by the variation of a single nucleotide at a specific location in the genome, including base conversion, transposition, deletion, and insertion. Following human genome sequencing, SNP detection and identification are important indicators for determining patient disease susceptibility and individual drug delivery.

Single nucleotide polymorphism (SNP)

The global market size of SNP genotyping is estimated to be $6.3B in 2028, according to the SNP genotyping market capacity analysis from Global Info Research (GIR). SNP detection is mainly applied in tumor susceptibility gene detection, targeted drug-gene detection, drug metabolism, and personalized medicine. To meet the needs of the market, SNP detection technologies continue to advance.

SNP genotyping detection techniques

SNP genotyping detection techniques include sequencing, qPCR, chip detection, and various novel PCR or qPCR based technologies, such as amplification refractory mutation system PCR (ARMS-PCR), andkompetitive allele-specific PCR (KASP).

1. Sequencing

Sanger sequencing is the “gold standard” for SNP detection, with the SNP detection rate being close to 100%. ,Sanger sequencing can also find unknown SNP loci and determine the mutation type and mutation location. It is the most direct and accurate SNP detection method.

Sanger sequencing principle

Genome-scale, high-throughput sequencing techniques can detect millions of single nucleotide polymorphisms. For example, these techniques can be used in genome-wide association studies (GWAS) to identify traits of interest that control genes or quantitative trait loci (QTLS) and are widely used in plant breeding and other fields.

2. qPCR

qPCR is one of the most common SNP genotyping methods, which is rapid, highly accurate, and cost-effective. Multiple analyses can be performed through qPCR, reducing analysis efforts and saving material costs by omitting PCR post-processing. qPCR based on the TaqMan probe is the most widely used SNP detection method in clinical diagnosis. The qPCR probe is perfectly matched to the template, and as the PCR reaction proceeds, the DNA polymerase hydrolyzes the 5′ reporter group as it extends to the probe position, releasing the fluorophore and generating a strong fluorescent signal. The SNP alleles in the corresponding samples are then determined by analyzing the different fluorescence detected.

However, the design and optimization of good probes are complex and challenging. There are currently several common probes on the market, such as MGB probes and molecular beacon probes. MGB probes can improve the specificity of SNP recognition by using MGB modifications in the TaqMan probe. Whereas, molecular beacon probes are a double-labeled stem-loop probe with a hairpin structure with SNP sites in the loop region. When a molecular beacon probe hybridizes with the template to form an extended state, the space between the fluorophore group and the quenched group becomes larger, and the fluorescence signal can be detected by the instrument. From there, the SNP can be determined. The advantage of the molecular beacon probe is that it can bind to the target region over a wide temperature range. Also, the shorter the loop length, the better the probe recognition.

Three types of qPCR probes

3. Chip Detection

Chip technology is a very powerful tool for gene analysis, which can carry out high-throughput large-scaled parallel information processing and functional research. This technology is mainly divided into oligonucleotide chips and DNA microarrays. When applied to SNP detection, the principle is that the probes are regularly arranged and fixed on the supporting objects (such as membrane, silicon chip, glass chip) by solid-phase in situ synthesis technology. After PCR amplification and fluorescent labeling, the sample DNA will be hybridized with the chip. Then, after the non-target fragment is removed, the fluorescence signal on the chip can be detected by scanning to determine the SNP site.

Chip detection

Conclusion

Each detection method has its advantages and disadvantages. According to different research directions and purposes, researchers can choose suitable SNP detection methods. How to evaluate SNP detection methods depends on the following three aspects:
• Design flexibility and success rate
• Allele invocation and allele identification efficiency
• Ease of running experiments and cost per sample

About Us

Synbio Technologies is committed to providing professional and efficient comprehensive DNA solutions for researchers, scientists, and drug developers around the globe. Our professional technical team, advanced equipment, and rational research strategies, offer you customized solutions help you carry out SNP genotype analysis, function identification, and other downstream applications.

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Reference:

[1] Ayalew H, Tsang PW, Chu C, Wang J, Liu S, Chen C, Ma XF. Comparison of TaqMan, KASP and rhAmp SNP genotyping platforms in hexaploid wheat. PLoS One. 2019 May 22; 14(5): e0217222.[2] Li B, Liu Y, Hao X, Dong J, Chen L, Li H, Wu W, Liu Y, Wang J, Wang Y, Li P. Universal probe-based intermediate primer-triggered qPCR (UPIP-qPCR) for SNP genotyping. BMC Genomics. 2021 Nov 24; 22(1): 850.[3] S S, Fuke S, Nagasawa H, Tsukahara T. Single nucleotide recognition using a probes-on-carrier DNA chip. Biotechniques. 2019 Feb; 66(2): 73-78.