Technical Principle and Methods Applied in SNP Genotyping
SNP, single nucleotide polymorphism, is caused by the change of single nucleotide. Generally, only two alleles can be found in an SNP locus, because of which, it is also called the biallelic gene. SNPs occur frequently in the human genome, with an average of one polymorphic locus per 1000 bases. Some SNP loci also affect the function of genes, leading to changes in biological traits and even disease.
Single nucleotide polymorphism is an important basis for studying the genetic variation of human families or animal and plant lines, which is therefore widely used in population genetics research (such as the origin, evolution, and migration of organisms) and disease-related genes. It plays an important role in pharmacogenomics, diagnostics, and biomedical research. In studying genome, it is the first and primary step to know the types of a genome. SNP genotyping is a kind of technique applying SNP to inspect gene types.
Firstly, the genomic fragment containing the SNP is amplified by PCR; afterward a single base extension is achieved by sequence-specific primers, and then the sample analyte is co-crystallized with the chip matrix and excited by a transient nanosecond (10–9 s) intense laser in a vacuum tube. The nucleotide molecule is thus desorbed into a singly charged ion. Since the ion flight time in the electric field is inversely proportional to the ion mass, the exact molecular weight of the sample analyte is obtained by detecting the flight time of the nucleotide molecule in the vacuum tube, thereby detecting the SNP locus information.
Methods about SNP genotyping
1 TaqMan Probe: PCR primers and TaqMan probes were designed for different SNP loci on the chromosome for real-time fluorescent PCR amplification. The 5'-end and 3'-end of the probe are labeled with a reporter fluorophore and a quenched fluorophore, respectively. When a PCR product is present in the solution, the probe anneals to the template, producing a substrate suitable for exonuclease activity, thereby cleaving the fluorescent molecule attached to the 5'-end of the probe from the probe, destroying both The PRET and the fluorescent molecules fluoresces. Usually used for small SNP site analysis.
2 SNaPshot: This technology is based on the principle of fluorescent labeling single base extension, also known as small sequencing, mainly for medium-flux SNP classification projects. In a reaction system containing a sequencing enzyme, four fluorescently labeled ddNTPs, different length extension primers and a PCR product template immediately adjacent to the 5'-end of the polymorphic site, the primer is terminated by one base extension and detected by the ABI sequencer. The SNP locus corresponding to the extension product is determined according to the position of the movement of the peak, and the type of the base to be incorporated is known according to the color of the peak, thereby determining the genotype of the sample. Templates for PCR products can be obtained by multiplex PCR reaction systems. Usually used for 10–30 SNP locus analysis.
3 HRM: High-resolution melting curve analysis (HRM) is an SNP research tool that has been developed in recent years to detect the presence or absence of SNPs and different SNPs by monitoring the binding of double-stranded DNA fluorescent dyes to PCR amplification products during heating. Whether the point is heterozygous or not affects the peak shape of the melting curve, so HRM analysis can effectively distinguish different SNP loci from different genotypes. This method of detection is not limited by the location and type of mutated bases. Without the need for sequence-specific probes, high-resolution melting can be performed directly after PCR to complete the analysis of the genotypes of the samples. This method eliminates the need to design probes, is simple, fast, low cost, accurate, and enables true closed-tube operation.
4 MassARRAY: molecular weight array technology is the world’s leading genetic analysis tool from Sequenom, which combines primer extension or cleavage reactions with sensitive and reliable MALDI-TOF-MS technology for genotyping. The iPLEX GOLD technology based on the MassARRAY platform can design PCR reactions and genotype detection up to 40 weights, with flexible experimental design and high accuracy of typing results. MassARRAY has the best price/performance ratio for tens to hundreds of SNP loci, depending on the application. It is especially suitable for validating the results found in genome-wide studies, or a limited number of study locus has determined the situation.
5 Illumina BeadXpress: Using Illumina’s BeadXpress system for batch SNP locus detection, 1–384 SNP loci can be detected simultaneously, often used for genomic chip results from confirmation, suitable for high-throughput detection. The microbead chip has high density, high reproducibility, high sensitivity, low sample loading, flexible customization, and high integration density, resulting in extremely high detection and screening speed, which can significantly reduce costs in high-throughput screening.
SNP genotyping service is widely available in the market. In genetic researches, asking for qualified genotyping service can guarantee a more convincible result which may push ahead ongoing studies.
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