Point mutations are not easily amplified mutation system (ARMS) analysis experiments
Point mutations are not easily amplified mutation system (ARMS) analysis experiments
The ARMS technique is based on the observation of the accessory oligonucleotide strand of a given DNA sequence, with the exception of a mismatch at the 3' end that does not function as a PCR primer under appropriate conditions. Because of the reliability of the ARMS assay, it is necessary to determine a combination of primer sequences and reaction conditions that will yield an ARMS product of a detectable allele when mispriming is minimized in non-target alleles. Conditions.
Operation method
Basic protocol ARMS test to detect single mutations
Materials and Instruments
Normal ARMS Primers Mutant ARMS Primers Common (Normal) Primers Move Basic protocol ARMS test to detect single mutations 1. Design normal, mutant, and common ARMS primers to fit the appropriate mutation according to the ARMS Primer Guidelines column that has been described. Select an appropriate reference primer from which all primers are obtained and prepare 50 umol/L of each solution. 2. Prepare normal and mutant ARMS reaction systems according to the guidelines in Table 9.7.2. Dispense 40 ul of pre-mixed reaction system into a reaction tube for one cycle and leave at room temperature for a few days or freeze at -20°C for a few months if necessary. 3. Add 5 W of prepared 10-50 ng/ul control DNA to each tube containing a mixture of N and M primers at room temperature. If possible, create separate controls containing DNA from normal, heterozygous, and pure individuals, including a blank control containing N and M tubes without added DNA. 4. Add a drop of paraffin oil to each tube and cap tightly, centrifuge at high speed in IOs. 5. Add 12.5 ul of 10XPCR reaction buffer to dilute Taq DNA polymerase and 5 ul of Taq DNA polymerase (5 U/ul is the final concentration) to 107.5 ul of sterile water (enough for 10 assays) and mix with a pipette gun. 6. Place the PCR tube in the thermocycling well and incubate at 94°C for 5 min, then take away and transfer to another tube with 5 ul Taq DNA polymerase for the shortest time, dilute to a low concentration and cover with a layer of paraffin oil, and place back into the 94°C blank well. Repeat this step until the enzyme is added to all tubes. 7. Stop the 94°C program and immediately run the following amplification program: 35 cycles: 1min 60°C (annealing) 1min 72°C (extension) 1 cycle 10min 72°C (extension) Store at room temperature for one week. If the PaKin-ElmerCetus9600 is used for rapid thermal cycling, its manufacturer requires that the number of cycles be changed to suit the characteristics of this type of machine. Upon completion of the amplification program, it is recommended that the temperature in the wells should be allowed to drop to 50°C after stopping the reaction to prevent the generation of artificial products. 8. Prepare 3% agarose gel using 4.5 g Nusieve':agarose 3:1 ratio, and 1 X TBE containing 0.5 ug/ul ethmoidin bromide buffer 150 ml (enough for 10 reactions). 9. Label a 0.5 ml microcentrifuge tube and add another 10 ul of Sampling Buffer to each tube. 10. Preferably in uncovered tubes in a laboratory with a vapor hood or isolation to avoid the presence of suspended particles of ARMS products, aspirate 25 ul of ARMS reactants from under the petroleum oil layer into the corresponding labeled tubes and mix well with a pipette gun. 11. 20 jul of product, including DNA molecular markers, were spotted in each lane of the gel, and electrophoresis was performed for 1-2 h at 120 V; imaging was performed under ultraviolet light. 12. Analyze the expected size of each lane and ARMS fragments according to the primers (A and B, C and D, or E and F) (for photographs the fragment size should be 360 bp, 813 bp, or 825 bp, respectively). There are 3 possible outcomes for normal and mutated ARMS products: Expected result. When the target allele is present in the sample, the target allele is missing if there is no visible product. Non-specific result: ARMS has product and the target allele is present in the sample; when the target allele is absent, the ARMS product is still visible. Non-sensitive result: ARMS product is missing or weak even if the target gene is present; no visible product when the allele is missing. (1) If the expected results are obtained from normal and mutant ARMS primers, then the optimized conditions are complete and the ARMS assay is ready for use - omit steps 13-14 and proceed to step 15. (2) If one or both ARMS primers are non-specific, proceed to step 13a. (3) If one or both ARMS primers are non-sensitive, proceed to step 13b. 13a. Enhance reaction specificity by repeating step 2 in the scheme with two reaction systems, each 2 to 5 times. 14a.If a nonspecific band is still observable, increase the length of the mismatch in this terminal base (Table 9.7.3) Repeat step 1. 13b. Repeat step 2 with two reaction systems to enhance sensitivity, using 2 to 4 times the number of ARMS primers in each of the two reaction mixtures or reducing the concentration of control primers. 14b.If the desired ARMS product band is still weak or missing, reduce the mismatch length in this terminal base and repeat step 1 according to Table 9.7.3. Changing an additional mismatch in an ARMS primer in a specific and sensitive ARMS reaction often causes a large change, changing the primer concentration has a small effect and optimizes the reaction. Subsequent optimization of the ARMS assay using DNA samples of known genotypes requires that large batches of ARMS reaction systems be prepared and then dispensed, and that the respective ARMS reaction systems be stable at -20°C for at least 6 months. 15. The information obtained from the optimized reaction conditions in the experimental manipulation goes to the analysis of the DNA samples; the samples are from steps 2 to 12. Include two blank (no DNA) and control (known genotype) samples, if possible, and refer to Table 9.7.4 if problems occur. It is often necessary to analyze the presence or absence of several different mutations in a particular gene, and one option would be to perform a series of individual ARMS assays; an alternative approach in this process would be to then perform multiple ARMS assays. Most of the variables in a single ARMS assay are normalized and primer sequences and concentrations are changed to obtain the desired results This approach is the same as for a single ARMS assay. The principle difference is that several pairs of mixed primers are optimized at the same time, increasing the complexity of the procedure. From the increased complexity it appears that it is not possible to generalize the procedure to the same level in the development of multiple ARMS assays. The basic steps of the procedure on the development of a single assay are the same, with the following differences. 1. In general, fewer unstable mismatches are used in the multiple ARMS assay compared to the single ARMS assay, using Table 9.7.1 (selection of mismatches) and Table 9.7.3 (length of mismatches) in order to select fewer unstable mismatches. 2. There is a fundamental size difference between the different ARMS products in the reaction. It is best to design ARMS products with >50bp difference in size. In the same experiment it is necessary to use a control for PCR amplification of a larger fragment of the product; two alternative procedures are listed in the oligonucleotide chain primers (see control primer recipes in the Appendix). 3. Combine several ARMS reactions into a twofold reaction system. Remove the normal and mutated reactions, then the two multiplex reactions contain some reaction systems and other mutated ARMS primers and normal ARMS primers. 4. For the detection of autosomal recessive disease genes, PCR control reactions can be omitted if at least four mutations are analyzed simultaneously and the two reaction tubes contain at least two normal-specific ARMS reactions. 5. It is possible to use a single common primer from a number of ARMS primers if the multiplex is for several mutations in the same exon neighboring positions. 6. The multiplex procedure is as important as a single ARMS assay, except that it may be necessary to repeat the entire procedure (steps 1-15) because several amplification reaction systems are being optimized at the same time. The following procedure describes a rapid extraction method for DNA from oral and blood samples to provide DNA suitable for the ARMS reaction conditions described in this unit. 1a. Add 800 ul of freshly prepared 170 mmol/L Edrophonium Chloride to a 1.5 ml centrifuge tube containing 200 ul of blood, mix on a shaker for 20 min, and centrifuge at high speed for 2 min to obtain a cell pellet, and remove the supernatant. 2b. Wash the cell pellet with 300 ul of 10 mmol/L NaCl/lO mmol/L EDTA and centrifuge at high speed for 15 s. Repeat the wash 3 times to remove visible hemoglobin. 1b. Vigorously shake 10 ml of 4% (m/v) sucrose water in the mouth for 20 s to produce a suspension containing epithelial cells. Collect the suspension in a 25 ml sterile plastic sample tube. Centrifuge at 1200 g for 10 min at room temperature to obtain cells. Remove the supernatant. To avoid any potential contamination problems, it is required that food and beverages be withheld for 30 min prior to collection of the epithelial cell suspension. 2b. Add 500ul of 10 mmol/L NaCl/lO mmol/L EDTA to resuspend the cells and transfer to a centrifuge tube. Centrifuge at high speed for 15s and remove the supernatant. 3. Resuspend the cell pellet with 500ul of 50 mmol/L NaCl/lO mmol/L EDTA and spin the Ios to resuspend the leukocyte pellet. Incubate in sterile water for 20 min. 4. Neutralize with 100 ul of 1 mol/L Tris-Cl and spin for 5s. 5. Centrifuge at high speed for 15s to remove cell debris. Store the supernatant (5 ul containing ~100 ug DNA, enough for one ARMS reaction) at -20°C until use. For more product details, please visit Aladdin Scientific website.
4dNTP 10 X PCR reaction buffer DNA template of known genotype Light mineral oil Taq DNA polymerase loading buffer DNA molecular size marker DNA sample
Perkin-Elmer Cetus thermal cyler (480 or TC) and appropriate reaction tubes
1min 94°C (denaturation)
