The principle of SNP genotyping technology is PCR amplification of genomic fragments containing SNP. The main features are high accuracy, strong flexibility and high throughput. The main method is the TaqMan probe method.

Technical principles

SNP genotyping

First, the SNP-containing genomic fragments are amplified by PCR, and then single-base extension is achieved by sequence-specific primers. Then, the sample analyte and the chip matrix are co-crystallized and then excited by an intense nanosecond (10-9s) intense laser in a vacuum tube. Nucleic acid molecules are desorbed into single-charged ions. Because the flight time of ions in the electric field is inversely proportional to the ion mass, the precise molecular weight of the sample analyte is obtained by detecting the flight time of the nucleic acid molecules in the vacuum tube, thereby detecting SNP site information.

Main features

The accuracy of SNP detection achieved by time-of-flight mass spectrometry (MALDI-TOF) can reach 99.9%. In addition to the advantages of high accuracy, flexibility, high throughput, and short detection cycle, the most attractive should be its cost performance. The time-of-flight mass spectrometry platform (MALDI-TOF) is an internationally-used research platform for genetic single nucleotide polymorphism (SNP). This method has become a new standard in this field with its scientificity and accuracy.

Main methods

1. TaqMan probe method

Design PCR primers and TaqMan probes for different SNP sites on chromosomes respectively, and perform real-time fluorescent PCR amplification. The 5'-end and 3'-end of the probe are labeled with a reporter fluorescent group and a quenching fluorescent group, respectively. When the PCR product is present in the solution, the probe is annealed to the template, which produces a substrate suitable for exonuclease activity, thereby cleaving the fluorescent molecule attached to the 5'-end of the probe from the probe, destroying the two PRET between fluorescent molecules emits fluorescence. Usually used for small SNP loci analysis.

2. SNaPshot method  

This technology was developed by American Applied Biology Company (ABI), and it is a typing technology based on the principle of single base extension of fluorescent labeling, also known as small sequencing, which is mainly aimed at medium-throughput SNP typing projects. In a reaction system containing sequencing enzymes, four fluorescently labeled ddNTPs, different length extension primers and PCR product templates immediately adjacent to the 5'-end of the polymorphic site, the primer extension is terminated by one base, and after detection by the ABI sequencer, The SNP site corresponding to the extension product is determined according to the shifting position of the peak, and the type of the incorporated base can be known according to the color of the peak, thereby determining the genotype of the sample. The PCR product template can be obtained by multiple PCR reaction system. Usually used for 10-30 SNP loci analysis.

3. The HRM method

high-resolution melting curve analysis (HRM) is a SNP research tool that has emerged in recent years. It detects the presence of SNPs by monitoring the combination of double-stranded DNA fluorescent dyes and PCR amplification products during real-time heating. Moreover, different SNP sites and whether they are heterozygotes will affect the peak shape of the melting curve, so HRM analysis can effectively distinguish between different SNP sites and different genotypes. This detection method is not limited by the location and type of the mutated base. No sequence-specific probes are required. After the PCR is completed, high-resolution melting is run directly to complete the analysis of the sample genotype. The method does not need to design a probe, and the operation is simple, fast, low in cost, accurate in result, and realizes the real closed tube operation.

4. Mass Array method  MassARRAY molecular weight array technology is the world's leading gene analysis tool launched by Sequenom. It combines primer extension or cleavage reaction with sensitive and reliable MALDI-TOF-MS technology to achieve genotyping detection. The iPLEX GOLD technology based on the MassARRAY platform can design up to 40-fold PCR reaction and genotype detection, with flexible experimental design and high accuracy of typing results. According to the needs of the application, when testing hundreds to thousands of samples from dozens to hundreds of SNP sites, MassARRAY has the best cost performance, especially suitable for verifying the results of genome-wide research findings, or a limited number of the situation where the research site has been determined.

5. Illumina BeadXpress method uses Illumina's BeadXpress system for batch SNP site detection, which can detect 1-384 SNP sites at the same time. It is often used to confirm the results of genomic chips and is suitable for high-throughput detection. The microbead chip has the characteristics of high density, high repeatability, high sensitivity, low sample load, flexible customization, etc., and extremely high integration density, thus obtaining extremely high detection screening speed, and can significantly reduce costs during high-throughput screening.

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CD Genomics