Single Nucleotide Polymorphism (SNP) Assay

Summary

SNP (Single Nucleotide Polymorphism), or Single Nucleotide Polymorphism, can be used to detect nucleic acid sequence polymorphisms (Polymorphism) due to changes in a single nucleotide.

Operation method

basic program

Principle

Nucleic acid sequence polymorphism (Polymorphism) due to a single nucleotide change. It is estimated that there is approximately one SNP per thousand bases in the human genome, which is much more extensive than either restriction fragment length polymorphism (RFLP) analysis or microsatellite markers (STR).SNPs are the smallest unit of genetic variation we can examine, and it has been estimated that there are approximately 10 million SNP loci in all human populations. It is generally believed that neighboring SNPs tend to be inherited together to the offspring. Thus, a group of associated SNP alleles located in a region of a chromosome is called a haplotype. Most chromosomal regions have only a few common haplotypes (each with a frequency of at least 5%), and they represent the majority of polymorphisms between people in a population. A chromosomal region can have many SNP loci, but once we have the haplotypes for the region, we can use only a few tagSNPs (tagSNPs) to genotype for most of the genetic polymorphic patterns.

Materials and Instruments

Single Nucleotide Polymorphism (SNP) Assay
Liquid nitrogen PBS GA buffer GB buffer Proteinase K Anhydrous ethanol Protein solution Rinsing solution
Centrifuge Tubes Centrifuge Waste Collection Tubes Adsorption Columns Water Baths Spectrophotometers Cryogenic Refrigerators

Move

I. DNA Extraction1. Take about 100 mg of fresh muscle tissue, rinse it well in PBS, place it in a 1.5 ml centrifuge tube, add liquid nitrogen and grind it rapidly.
2. Add 200 ul buffer GA, shake until thoroughly suspended. Add 20 ul proteinase K (20 mg/ml) solution and mix well.
3. Add 220 ul buffer GB, mix thoroughly and digest overnight at 37°C. The solution becomes clear. Add 220 ul anhydrous ethanol and mix thoroughly, at which point a flocculent precipitate may appear.
4. Add both the solution obtained in the above step and the flocculent precipitate to an adsorbent column CB (the adsorbent column is placed in the waste collection tube) and centrifuge at 12,000 rpm for 30 seconds, discard the waste solution.
5. Add 500 ul of Deproteinizing Solution GD (check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 30 seconds, and discard the waste solution.
6. Add 700 ul of Rinse Solution GW (check that anhydrous ethanol has been added before use), centrifuge at 12,000 rpm for 30 seconds and discard the waste solution. Add 500 ul of Rinse Solution GW, centrifuge at 12,000 rpm for 30 seconds and discard the waste solution. Return the column CB to the waste collection tube and centrifuge at 12,000 rpm for 2 minutes to remove as much of the rinse solution as possible.
7. Transfer the Adsorbent Column CB to a clean centrifuge tube, add 100 ul of Elution Buffer (Elution Buffer should be pre-warmed in a water bath at 60-70°C), mix well, and allow to stand at room temperature for 15 minutes before centrifuging at 12,000 rpm for 30 seconds. For the second elution, add 50 ul of Elution Buffer to the column and leave at room temperature for 15 minutes, centrifuge at 12,000 rpm for 30 seconds.
8. The concentration of the extracted genomic DNA was measured using a Beckman DU 640 spectrophotometer with a significant absorption peak at _OD260. The purity of the extracted genomic DNA was also checked and the _OD260/280 value should be 1.7-1.9.
9. Remove the corresponding volume of DNA solution from the stock solution and dilute it to 50 ng/ul. Store the stock solution at -70℃ and the diluted solution at -20℃.
PCR amplification of target fragments
1. Prepare the reaction system in a standard reaction tube according to the relevant reagent instructions, a typical PCR reaction system is as follows (20 ul system):
2. Pull the handle on the instrument lid to the left to remove the lid, carefully place the sample tube in the corresponding sample hole of the instrument, gently close the lid, and slowly tighten the knob at the top so that the hot lid is in close contact with the sample tube and the sample is placed.
III. Edit a program on the T1 PCR instrument
1. Press [C programs] to enter the editing mode. To create a program in the main directory press [D enter]. To enter a subdirectory, use the → key to move the cursor to the right, then use the ↑↓ keys to select a subdirectory. Press [D enter] to enter the selected subdirectory.
2. Enter the temperature requested in the program: Confirm the temperature with [D enter]. Enter the time for it, spacing it with decimal points. The sequence is h.m.s. Confirm the time setting with [D enter] or move to the next field with the cursor keys. The # indicates the number of cycles. Setting cycle value = total cycle value - 1, i.e., "29" should be entered when the total number of cycles is 30. Use [C pgm ok] to store a complete program. The program data is permanently stored in memory.
Running the program
Press [B start/stop] to select a program. Use the →↑↓ keys to select a subdirectory, or use [D enter] to enter the main directory. Enter the number of the program you wish to start. Alternatively, press [A list] to select a program from the list of all programs in that subdirectory. Use the ↑↓ keys to scroll through the list to select it. Confirm the program highlighted with a strong light with [D enter]. Press [D start] to start the program.
v. controlling the test process
During the run, press the A button to get information about the time remaining in the program. When the run is complete, press the STOP button to terminate the experiment and press YES to confirm termination. Carefully unscrew the hot cover, follow the operation sequence of placing the samples, open the cover, take out the experimental samples, then cover the cover and turn off the power, this experiment is finished.
VI. PCR Product Sequencing
Completed by a service company specializing in sequencing.
VII. Data analysis
A small amount can be read manually, and a large amount can be read by software. Compare the position of SNPs found in the genome: focus on promoter region, exon region (including cSNP in coding region and 5' and 3' UTR), shear boundary, etc., and whether codon changes lead to amino acid changes: missense mutation, nonsense mutation, termination mutation.

Caveat

1. In order to ensure the authenticity and reliability of the sequencing of the target region to be tested, the primers should be designed so that the boundary of the target region to be tested is at least 50 bp away from the upstream and downstream primers;2. Primer design is recommended to use online method to ensure the success rate;3. To ensure the sensitivity of sequencing, the fragment size of PCR products should be in the range of 250 bp-650 bp;4. for the convenience of the experiment, it is recommended that primers be synthesized into 1 o.d/tube to facilitate the separation of primers for PCR and sequencing;5. to ensure the specificity of the primers, it is recommended that primer design be confirmed by blast on NCBI;6. to prevent degradation, PCR products should be sequenced as soon as possible, otherwise they should be stored at -20℃ and not for too long;7. to ensure the authenticity of the results, it is recommended to confirm the key points by reverse sequencing.

Common Problems

The characteristics of SNPs themselves determine that they are more suitable for research on genetic dissection of complex traits and diseases as well as population-based gene identification:

1. SNPs are numerous and widely distributed. It is estimated that there is one SNP for every 1,000 nucleotides in the human genome, and there are more than 3 million SNPs in 3 billion bases in humans.SNPs are spread throughout the human genome, and according to the position of SNPs in genes, they can be divided into gene coding-region SNPs (cSNPs), gene perigenic SNPs ( Coding-region SNPs (cSNPs), perigenic SNPs (pSNPs), and intergenic SNPs (iSNPs).

2. SNPs are suitable for rapid and large-scale screening. Although there are four types of bases that make up DNA, SNPs are generally composed of only two bases, so it is a dimorphic marker, i.e., a biallelic (two alleles). Because of the dimorphism of SNP, either/or, SNPs in genomic screening often only need to +/- analysis, without analyzing the length of the fragment, which facilitates the development of automated technology screening or detection of SNPs.

3. Easy estimation of SNP allele frequencies. Estimation of allele frequency using mixed samples is an efficient and fast strategy. The principle of this strategy is: first select the reference sample to make a standard curve, and then compare the mixed samples to be tested with the standard curve, and then determine the frequencies of various alleles in the mixed samples according to the proportion of the resulting signals.

The dimorphism of SNPs also facilitates the genotyping of SNPs. Genotyping of SNPs includes three aspects: (1) the chemical reaction used to identify the genotype, commonly used techniques include: DNA molecular hybridization, primer extension, allele-specific oligonucleotide linkage reaction, flanking probe cutting reaction, as well as adaptive techniques based on these methods; (2) the mode used to complete these chemical reactions, including liquid-phase reactions, reactions carried out on the solid-phase support, and both of them. reactions, and reactions performed on both. (3) At the end of the chemical reaction, a biotechnology system needs to be applied to detect the results of the reaction.

Source: Gao Jinrong, Molecular Biology, Chemical Industry Press, 2011.

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