Quantitative methylation-specific PCR
Quantitative methylation-specific PCR
Quantitative methylation specific PCR (qMSP) is a molecular biology method for detecting the degree of DNA methylation, which can determine the percentage of DNA site-specific methylation status, and is suitable for describing the role of methylation in gene expression as well as the molecular biology mechanism of disease development. It is suitable for describing the role of methylation in gene expression and the molecular biological mechanisms of disease development.
Principle
Quantitative methylation-specific PCR is a PCR-based technique that combines specific restriction endonucleases and fluorescent quantitative PCR to accurately characterize the extent of DNA methylation by determining the proportion of PCR products that are synthesized from methylated versus unmethylated DNA-specific loci.
Appliance
Quantitative methylation-specific PCR can be used to detect and quantify the role of DNA methylation in gene expression, genome stability, cancer and other diseases.
Operation method
Quantitative methylation-specific PCR (qMSP)
Principle
Quantitative methylation-specific PCR is a PCR-based technique that combines specific restriction endonucleases and fluorescent quantitative PCR to accurately characterize the extent of DNA methylation by determining the proportion of PCR products that are synthesized from methylated versus unmethylated DNA-specific loci.
Materials and Instruments
① PCR kit: the reaction system contains dNTPs, Taq DNA polymerase, MgCl Move The procedure may be different for different manufacturers and models of Quantitative Methylation Specific PCR kits, and the general procedure is given here as a reference: a. Extract the DNA of the sample to be tested and digest the RNA. b. Check the purity and concentration of DNA to ensure the accuracy of PCR reaction. 2. a. According to the experimental needs, choose the appropriate methylation detection methods, including methylation-specific PCR or methylation-sensitive restriction endonuclease (MS-RE) digestion. b. Methylation or non-methylation of the samples to be tested, as required. 3. a. Configure the PCR reaction system according to the instructions in the kit. b. Add the DNA template to the PCR system. b. Add the DNA template into the PCR tube. 4. a. Set the PCR parameters according to the requirements of the kit. b. At the end of the PCR reaction, the size and quantity of PCR products can be checked by running a gel. 5. a. PCR products are analyzed to determine the size and methylation status of the DNA fragments. b. The data are analyzed statistically. b. The data are analyzed statistically to determine the degree of methylation. Caveat ① Avoid contamination in all operations before handling DNA to ensure the reliability of experimental data.② Pay attention to the storage of consumables to avoid the influence of ambient temperature.③ Follow the instructions of the kit when making the PCR reaction system. Any change will affect the PCR reaction system and lead to inaccurate final data.④ The methylation-specific PCR system needs to be optimized, so care should be taken for sample purification.⑤ In the PCR reaction, two sets of methylated and non-methylated primers need to be amplified separately in order to compare their PCR products and to determine the site of methylation.(6) The location of methylation and the distance to the chimeric site affect the effectiveness of the specific primers and should be designed accordingly.(vii) To ensure the accuracy of the results, validation experiments are required to verify the methylation status and restriction modifications of different DNA samples. Common Problems ① Why are samples labeled with molecular markers for the PCR reaction system? A: Molecular markers are used to detect and verify PCR reaction products and samples. ② Why is it possible to obtain a specific fragment of the amplification product within 20-40 cycles after the PCR reaction? A: The PCR reaction is a template-free amplification method, the reaction product is specified to be short and long, the reaction mode is exponential, and the reaction period is related to the specifics of the PCR system in the kit. ③ What should I do if the PCR reaction yields an inconspicuous enhancement band? A: Check and optimize the reaction system of the product, you can increase the difference between the two concentrations, increase the reaction temperature, and change the primers. ④ Too little amplification product A: The quality of the sample is not good, suggest to increase the concentration of DNA template. A: Too much template DNA, reduce the amount of template DNA gradually. A: It may be due to expired primers or restriction enzyme digestion. Check the quality of primers and the reasonableness of the restriction enzyme digestion reaction system. Check the quality of the primers and the restriction enzyme digestion system: 1. Frommer M, McDonald LE, Millar DS et al. "A genome sequencing protocol that produces a positive display of 5-methylcytosine residues in individual DNA strands". Proceedings of the National Academy of Sciences of the United States of America, 1992, 89: pp. 1827-1831. 2. Herman JG, Graff JR, et al. "Methylation-specific PCR: a novel PCR-based method for methylation status analysis". Proceedings of the National Academy of Sciences of the United States of America, 1996, 93: pp. 9821-9826. 3. Eads CA, Danenberg KD, Kawakami K, et al. "'MethyLight': a high-throughput assay for measuring DNA methylation". Nucleic Acids Research, 2000, 28(8): p. E32. For more product details, please visit Aladdin Scientific website.
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MgCl2 and PCR reaction buffer.
② DNA purification kit
③ Restriction endonucleases: e.g. HhaI, etc. ④ Template DNA: the DNA to be tested.
③ Restriction endonucleases: e.g. HhaI, etc. ④ Template DNA: DNA sample to be tested
⑤ Methylation-specific primers: methylation primers and non-methylation primers (two groups)
⑤ Methylation-specific primers: methylation primers and non-methylation primers (two sets) ⑥ Taqman probe
⑦ DEPC water: used for PCR gradient dilution to generate a standard curve.
⑧ PCR instrument
⑧ PCR instrument ⑨ Printer and computer
1. DNA template preparation:
2. Methylation status detection:
3. PCR reaction system configuration:
4. PCR reaction conditions:
5. Data analysis: a. PCR products are analyzed:
⑤ Too much amplification product
(6) Too little PCR amplification product.