Arabidopsis belongs to the Angiosperms, Dicots, Cruciferae, and is a biennial herb. SNP marker technology has unique advantages. It is more direct for comparing different genotypes, and therefore more suitable for genetic analysis of complex shapes and identification of genes that cause population differences. It plays a very important role in the construction of high-density genetic linkage maps of plants, cloning of new genes, and SNP marker-assisted breeding.
Single Nucleotide Polymorphism (SNPs) refers to DNA sequence polymorphism caused by changes such as conversion, transversion, insertion, and deletion at a single nucleotide position at the genome level. In a population, when two different nucleotides exist at a specific nucleotide position in the genome and the frequency of occurrence is greater than 1% (some people also propose 2%), it is considered a SNP. Due to the large number of such landmarks and the high coverage density, the accuracy of genome mapping and gene positioning can be greatly improved.
The detection of SNPs was initially based on DNA sequencing using gel electrophoresis. In recent years, some semi-automatic or fully automatic methods for identifying and detecting a large number of SNPs are being developed. The current experimental methods include restriction fragment length polymorphism (RFLP) based on gel electrophoresis, allele-specific oligonucleotide (ASO) hybridization, single-strand conformation polymorphism (SSCP) analysis, Taqman method based on fluorescence resonance energy transfer and DNA chip method, mass spectrometry, microsequencing, etc. Denaturing high performance liquid chromatography (DHPLC) is a highly efficient and rapid SNP screening technology developed later, which has attracted much attention due to its high sensitivity, low cost and fully automated operation.
In this experiment, polyacrylamide electrophoresis was used to observe the SNP of a gene fragment in different ecotypes of Arabidopsis. According to the sequencing results, the relative molecular mass of the At5962130 fragments of different ecotypes of Arabidopsis, Ws and Col, was the same, but there was a difference in a single site, which could not be distinguished by conventional agarose gel electrophoresis. We first amplified the At5962130 fragment of Arabidopsis by PCR, and then denatured and renatured the PCR products and separated them by polyacrylamide electrophoresis. Due to the presence of SNPs, the At5962130 amplified fragments in Ws and Col can be distinguished by the difference in their swimming speed due to their different conformations. For the F1 generation (heterozygous) of the hybridization of Ws and Col, the PCR product must contain two different DNAs with a single site difference, and the ratio of the two is 1:1. Denaturing and renaturing the PCR products will form homologous duplexes and heteroduplexes. Therefore, after polyacrylamide electrophoresis, the three products with different conformations can be distinguished.
For DNA extraction and purification methods, please refer to extraction and purification of rice genomic DNA.
The Arabidopsis gene At5962130 fragment was amplified by PCR, and the reaction system is shown in Table 1.
The template was genomic DNA of three seedlings of different ecotypes of Arabidopsis, CO, Ws and CO, Ws hybrid F1.
PCR reaction program: 94°C, 5 min; 94°C, 1 min; 56°C, 1 min; 72°C, 1 min, a total of 28 cycles; 72°C, 15 min. The length of the PCR product is 1057 bp.
Table 1. PCR Conditions
| Reagents | Sample Volume/µL |
|---|---|
| Upper and lower primers (10 µmol/L) | 0.5+0.5 |
| 10×PCR buffer | 2 |
| dNTP | 2 |
| Taq enzyme | 0.2 |
| H2O | 13.8 |
| Template | 1 |
| Total | 20 |
Take 10 µL of the PCR product, cover it with paraffin oil, denature it at 94℃ for 5 min, and renature it at 42℃ for 30 min.
Polyacrylamide (14%) gel electrophoresis was performed at 300 V for 1.5 h. The electrophoresis buffer was 0.5× TBE.
After electrophoresis, the gel was stained with TBE solution of 0.5 µg/mL EB for 15 min, and the bands were observed under UV light.
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