8 detection methods for single nucleotide variation (SNP)!

Sanger sequencing is a classic method of DNA sequence analysis, which can directly obtain nucleic acid sequence information and is the "gold standard" for SNP detection. Moreover, Sanger sequencing can discover unknown SNP sites and determine the mutation type and mutation position of the SNP. It is the most direct and accurate SNP detection method that cannot be replaced.

When using the sequencing method to detect SNP, firstly, the target sequence containing the SNP site can be amplified by PCR to form a DNA fragment, and then the nucleic acid sequence of the target region can be obtained by Sanger sequencing, and the SNP site can be compared. Determine if there are mutation sites.

Sanger sequencing is based on dideoxyribonucleotide (ddNTP) terminal termination method for detection. That is, in four separate reaction systems, four kinds of A, T, G, and C dideoxynucleotides with different color marks are correspondingly incorporated, so that the nucleotides start to elongate at a fixed point, and the elongation If ddNTP is incorporated during the process, the extension cannot continue due to the lack of 3'-OH on the base, so it will randomly stop at a specific base, forming a series of nucleic acid fragments of different lengths with a difference of one base, and then use capillary electrophoresis These nucleic acid fragments of different lengths are separated, and finally the base sequence of the nucleic acid to be tested is read through the colors of different base markers, thereby obtaining the nucleotide sequence of the target region.

The TaqMan probe is a double-labeled, self-quenching hydrolysis probe. Its 5' and 3' ends are respectively labeled with a fluorescent group and a quencher group. When the probe structure is complete, the distance between the two is relatively close, and the fluorescent group signal can be quenched. In the process of PCR amplification, if the TaqMan probe completely matches the target sequence, the probe can be combined on the DNA template. At this time, when the Taq enzyme extends to the probe position, its exonuclease activity will cut the hydrolyzed probe, The fluorophore is released, resulting in an enhanced fluorescent signal. Moreover, with the increase of the amplification product, the fluorescence signal becomes stronger and stronger, so that the change process of the fluorescence signal can be monitored in real time by the instrument.

For biallelic SNPs, two corresponding probes can be designed respectively. Only when the probes are completely matched with the template, the probes can be hydrolyzed during the amplification process to generate a strong fluorescent signal. If there is a mismatch between the target sequences, the fluorescence intensity will be weakened, so the SNP site can be detected by the fluorescence intensity.

Since the length of the MGB probe can be designed to be shorter, it is beneficial to improve the recognition specificity of the probe, so the TaqMan probe used to detect SNP is often modified with MGB. However, during testing, more probes need to be screened, and the cost of probe synthesis is relatively high.

Amplification Refractory Mutation System PCR (Amplification Refractory Mutation System PCR, ARMS-PCR), also known as Allele-Specific PCR (Allele-Specific PCR, AS-PCR), is based on the fact that Taq DNA polymerase cannot repair the primer 3' A detection method in which a single base mismatch at the end prevents amplification. During the amplification process, only when the base at the 3' end of the primer is complementary to the allele at the SNP site, can the amplification be extended normally; When there is no complementary pairing, no amplification reaction occurs, and the amplified product is detected by gel electrophoresis or fluorescent PCR to determine the SNP genotype.

On the basis of ARMS-PCR, some improved methods have also been developed, such as tetra-primer amplification refractory mutation system PCR (tetra-primer amplification refractory mutation system PCR, Tetra-primer ARMS-PCR). This method designs four primers, two of which are inner primers whose 3' ends are located at the SNP site, and the two primers have opposite directions and belong to different genotypes; the other two are universal outer primers, and the two primers are compatible with the SNP The distance between the points should be different. In this way, during the reaction, if the four primers are all fully matched with the template, three products of different lengths can be amplified (the two internal primers are located at the same site, and no long fragment amplification product is formed); and if If one of the two internal primers does not match the template, only two products of different lengths will be formed. Therefore, the genotype can be judged according to the size of the product after electrophoresis.

However, due to the long detection time of gel electrophoresis and the possibility of pollution caused by opening the lid for product analysis, most of the products developed using the ARMS-PCR method on the market use real-time fluorescent PCR for closed-tube detection. In the fluorescent PCR detection of ARMS-PCR method, TaqMan probes are also used, but the SNP site is designed at the 3' end of the primer, so theoretically only when the 3' end of the primer matches the template completely, the amplification curve can be formed; but in In actual detection, a single base mismatch can still be extended and amplified, but the efficiency is low. In order to improve its specificity, it is sometimes necessary to artificially introduce mismatched bases near the 3' end of the primer to reduce the amplification efficiency of non-target sequences.

Molecular Beacon is a double-labeled oligonucleotide probe, its 5' end and 3' end are labeled with a fluorescent group and a quencher group respectively, and some bases at the 5' end and 3' end of the probe can be complementary , thus forming a stem-loop structure, so that the fluorescent group and the quencher group are close to each other and the fluorescent signal is low. The ring structure part of the molecular beacon contains the SNP detection site. When the template completely matches the nucleic acid sequence of the ring structure of the molecular beacon, the molecular beacon can hybridize with the template to form an extended state, resulting in a fluorescent group and a quencher group The spatial distance increases and the fluorescent signal increases, so it can be detected by the instrument and the SNP site can be determined.

By using different fluorophores to label molecular beacons, SNP types can be distinguished. This method is similar to the TaqMan probe method, which distinguishes SNPs by the recognition ability of a single base in the probe. It's just that the molecular beacon adopts a stem-loop structure, while the TaqMan probe is modified with MGB to improve the specificity of the probe for SNP recognition.

High-resolution melting analysis (high-resolution melting analysis, HRM) is a method for analysis and detection through the combination of specific dyes and amplified products to form specific melting peaks. The special material used is saturated fluorescent dyes, which have stronger DNA binding ability, and does not affect PCR amplification, and will not rearrange during DNA melting, making the melting curve have higher resolution.

The inherent characteristics of nucleic acid fragments, such as the length of the DNA sequence, GC content, and base complementarity differences, can all affect the high-resolution melting curve, and combined with a high-precision fluorescent quantitative PCR instrument, its resolution accuracy can reach a single base Differentiation of differences can be used to determine SNP loci.

SNaPshot is a commercial technology developed by Applied Biotechnology (ABI) in the United States. It is a typing technology based on the principle of single-base extension of fluorescent markers. This method is also called small sequencing. The key to its primer design is that the 3' end of the extension primer should be close to the SNP site, and different length extension primers can be designed for different SNP sites, so that the SNP sites can be distinguished by the length of the primers.

During detection, multiplex PCR amplification is carried out in a ddNTP system containing sequencing enzymes and four fluorescent labels. Since the added nucleotides are dideoxyribonucleotides, the primers are terminated after one base extension, and the extension products pass through Sequencer detection can determine the SNP site corresponding to the extension product according to the position of the peak movement, and the base type of the SNP site can be determined according to the color of the peak.

KASP (Kompetitive Allele-Specific PCR) is a method for detecting SNPs based on the specific matching of primer terminal bases. This method is different from conventional fluorescent PCR in the design of primers and probes. The corresponding upstream primers, the 3' ends of the primers are located at their respective SNP sites, and the 5' ends of the primers each have a unique tag sequence, while the downstream primer is a routinely designed shared primer; secondly, design two Fluorescent probes with the same label sequence as the upstream primer, the 5' ends of the probes are respectively labeled with different fluorescent groups, and corresponding quenching probes are designed corresponding to the fluorescent probes, and the 3' ends of the quenching probes are End-labeled quenching group. Therefore, when the amplification is not started, the fluorescent probe is combined with the quencher probe, and the fluorescent group and the quencher group are close to each other, so no fluorescent signal can be generated. When one or two of the designed upstream primers completely match the template, the amplification can be started, and the amplified product is then amplified in combination with the downstream primers to form a DNA fragment containing the tag sequence; The DNA fragment can be combined with a fluorescent probe, and the 3' end of the fluorescent probe can be used as a primer to initiate amplification, and finally a fluorescently labeled amplification product is formed, and the quenching probe corresponding to the fluorescent probe is activated during the amplification process. It is cleaved, so that the fluorescent signal of the amplification process is enhanced, and the SNP site can be detected.

The KASP method synthesizes two universal fluorescent probes and two universal quenching probes for the upstream primer label sequence, and then combines specific primers for different SNP sites to detect multiple SNP sites.