Methylation-specific PCR (MS-PCR) is a site-specific methylation detection technique, which is one of the most commonly used and highly accurate methods to study DNA methylation. Currently, there is one main method used for methylation-specific PCR: methylation-specific PCR.
Principle
The basic principle of methylation-specific PCR is to deaminate the unmethylated cytosine in DNA to uracil by bisulfite, while the methylated cytosine remains unchanged, and all uracil is converted to thymine by PCR amplification of the desired fragments, and finally, the PCR products are sequenced and compared with the untreated sequences to determine whether the CpG sites are methylated or not.
This method is a very reliable and accurate method, which can clarify the methylation status of each CpG site in the target fragment, but it requires sequencing of a large number of PCR products, and the process is more cumbersome and expensive. 1996 Herman et al. created a new method based on the use of bisulfite treatment, which treats the DNA with bisulfite first, so that unmethylated cytosine is converted to uracil, and methylated cytosine is converted to thymine, while the methylated uracil is converted to thymine. uracil, while the methylated ones remain unchanged, followed by primer-specific PCR (methylation-specific PCR, MS-PCR).
In MS-PCR, two pairs of primers are designed, and both pairs of primers are designed to end at the end of the detection site; each pair of primers can only be complementarily paired with methylated or non-methylated sequences after bisulfite treatment, i.e., one pair binds to the methylated DNA strand and the other pair binds to the non-methylated DNA strand after the treatment.
The MSP amplification product is detected, and if a fragment can be amplified with primers targeting the treated methylated DNA strand, it means that the detected site is methylated; if a fragment is amplified with primers targeting the treated non-methylated DNA strand, it means that the detected site is not methylated.
Appliance
Methylation-specific PCR is commonly used in the following areas: 1. DNA methylation and genetic imprinting, embryonic development DN methylation plays an extremely important role in maintaining normal cell function, genetic imprinting, and embryonic development. Studies have shown that the normal development of the embryo benefits from proper methylation of genomic DNA. For example, the absence of any of the methyltransferases is lethal to mouse embryo development. In addition, allelic repression is regulated by imprinting control regions (ICRs), which are methylated at one allele in both parents. Aberrant expression of imprinted genes can cause a variety of human diseases accompanied by mutations and phenotypic defects. For example, Prader-Willi/Angelman syndrome, etc. 2. DNA Methylation and Tumors Tumorigenesis has a multistage and multigenic character, and is the result of the combined action of polygenic genetics and epigenetics. Among the epigenetic mechanisms, DNA methylation has been studied the most and is one of the most deeply researched mechanisms. Accompanying tumorigenesis and progression, DN methylation abnormalities are detected in body fluids such as peripheral circulating serum, plasma and urine of tumor patients. Therefore, the use of DNA methylation analysis technology to detect the DNA methylation level of specific molecules in body fluids is a potential means for early diagnosis, disease monitoring and efficacy assessment of tumors, which is of great significance to the diagnosis and treatment of clinical tumors.
Operation method
Methylation-specific PCR
Principle
The basic principle of methylation-specific PCR is that after DNA is treated with bisulfite, unmethylated cytosine becomes uracil, while methylated cytosine remains unchanged, and the treated product is used as a template for specific amplification by adding methylation-specific primers (primer I) or non-methylation-specific primers (primer II), and only fragments that incorporate complete methylation or non-methylation specific primers can be amplified. Only fragments combining fully methylated or non-methylated specific primers can amplify the product.
Materials and Instruments
Equipment: Move The basic process of methylation-specific PCR can be divided into the following steps: The key to primer design is the design of specific primers. The primer sequence is designed in the cytosine-rich region to differentiate between the non-methylated DNA transformed by sodium bisulfite treatment and the non-transformed methylated DNA, and at least three CpG sites are included in the 3' end of the primer to ensure the differentiation between methylated and non-methylated DNA. Wild-type primer pairs are designed directly from the genome sequence to be tested. Methylated primer pairs and non-methylated primer pairs are designed based on the sequence of the CpG sites of the sequence to be tested after sodium bisulfite transformation. The wild-type primers can only amplify the gene fragments that have not been treated with sodium bisulfite, and the methylation primer pairs and non-methylation primer pairs can only amplify the methylated and non-methylated gene fragments, so as to achieve the purpose of detecting gene methylation.
① PCR instrument
② Centrifuge
Reagents
Reagents:
① Genomic DNA
② 3 mol/L NaOH solution
② 3 mol/L NaOH solution ③ 10 mmol/L, pH 5.0 Hydroquinone
④ 3 mol/L, pH 5.0 NaHSO
3
⑤ 3 mol/L NaAc
⑥ Cold ethanol
⑦ TE buffer
(1) Extraction of DNA from fresh mammalian tissues
① Cut about 5 g of tissue, remove connective tissue, absorbent paper to absorb the blood, cut into pieces and put into the mortar (the finer the better).
② Pour in liquid nitrogen, grind into powder, add 10 ml of separation buffer (separation buffer: 10 mmol/L Tris-Cl pH7.4, 10 mmol/L NaCl, 25 mmol/L EDTA).
③ Add 1 ml 10% SDS and mix well, at which time the sample becomes sticky.
④ Add 50 μl or 1 mg of protease K. Hold at 37 ℃ for 1~2 h until the tissue is completely disintegrated.
⑤ Add 1 ml of 5 mol/L NaCl, mix well, and centrifuge at 5 000 r/min for several seconds.
⑥ Take the supernatant in a new centrifuge tube and extract with equal volume of phenol-chloroform-isoamyl alcohol (25:24:1). After stratification, centrifuge at 3 000 r/min for 5 min.
(vii) Take the upper aqueous phase to a clean centrifuge tube and extract with 2 times the volume of ether (operated under ventilation).
(8) Remove the upper layer of ether and retain the lower aqueous phase.
⑨ Add 1/10 volume of 3 mol/L NaAc and 2 times volume of anhydrous ethanol and mix upside down to precipitate DNA. 10~20 min at room temperature, the DNA precipitate will form white flocs.
The DNA precipitates should be white and flocculent. ⑩ Hook out the DNA precipitates with a glass rod, rinse them in 70% ethanol, blot them on absorbent paper, dissolve them in 1 ml of TE, and store them at -20 ℃.
If there are insoluble particles in the DNA solution, centrifuge it briefly at 5,000 r/min and take the supernatant; if you want to remove the RNA, add 5 μl of RNaseA (10 μg/μl), keep it warm at 37 ℃ for 30 min, and extract it with phenol, then reprecipitate the DNA according to steps ⑨ and ⑩.
(2) Extraction of DNA from cells
① Discard the treated cells from the culture medium, wash them with PBS and digest them with trypsin.
② Add PBS and collect the cells in a 15 ml centrifuge tube.
② Add PBS and collect the cells in a 15 ml centrifuge tube. ③ Centrifuge at 1,000 r/min for 5 min.
③ Centrifuge at 1 000 r/min for 5 min. ④ Discard the supernatant, resuspend with PBS and transfer to a 1.5 ml centrifuge tube.
⑤ Centrifuge at 5000 r/min for 1 min, discard the supernatant, and dissolve the precipitate with 1 times the volume of lysis solution (containing 0.2 mg/ml protease K, 150 mmo/L NaCl, 40 mmol/L EDTA, 10 mo/L Tris-HCl, 1% SDS, pH 8.0).
⑥ Warm bath at 37 ℃ for 6 h. ⑦ Centrifugation (14 000 r/kg).
⑦ Centrifuge (14 000 r/min, 5 min, 4 ℃), take the aqueous phase, add an equal volume of phenol chloroform isoamyl alcohol (25:24:1). Shake gently, centrifuge at 10 000 r/min for 10 min, repeat twice.
⑧ Take the aqueous phase, add NaCl to make the final concentration of 140 mol/L and twice the volume of pre-cooled anhydrous ethanol mixing hook.
⑨ -20 ℃ overnight.
⑩ Centrifuge, wash with 70% ethanol, air dry, and add TE buffer containing RNAase to re-mix.
(iii) DNA modification by sodium bisulfite① Take 2 μg of genomic DNA and dilute to 50 μl with sterilized distilled water.
② Add 5.5 μl of freshly prepared 3 mol/L NaOH (final concentration 0.3 mol/L), and then take a water bath at 37 ℃ for 10 min to denature the DNA into single stranded DNA.
(iii) Add 30 μl of freshly prepared 10 mmol/L hydroquinone (hydroquinone) and 520 μl of 3 mol/L sodium bisulfite in turn, invert and mix gently, cover with 100 μl of paraffin oil to prevent evaporation of the liquid, and then protect from light for 16 h at 50 ℃ in a water bath.
(D) Purification of modified DNA① Preheat sterilized double-distilled water to 80 ℃.
② Use Promega DNA Clean Up (A7280) purification kit to purify DNA and follow the instructions.
③ Elute the DNA with 50 pl of sterile water and centrifuge at 12,000 r/min for 1 min.
④ Add 3 mol/L NaOH 5.5 μl (final concentration 0.3 mol/L), and terminate the modification at room temperature for 5 min.
⑤ Add 10 mol/L ammonium acetate 23 μl, neutralize, and precipitate with 3 times the volume of ice anhydrous ethanol at -20 ℃ for 4 h. ⑤ Add 10 mol/L ammonium acetate 23 μl.
⑥ Centrifuge (12 000 r/min, 15 min, 4 ℃), collect the precipitate, and dry it.
(vii) Resuspend in sterile water and freeze at -20 ℃.
(v) PCR reaction system and parametersReaction system (50 μl): 5 μl of 10 × PCR buffer, 2.5 μl of 25 mmol/L NTP mixture, 1 μl of forward primer (300 ng/μl), 1 μl of reverse primer (300 ng/μl), 2 μl of DNA template (less than 2 μg), 38.5 μl of dH2O, 1.25 U of Tag DNA polymerase.
Reaction parameters: 95 ℃ pre-denaturation for 5 min, add Taq DNA polymerase 1.25 U; 95 ℃ for 30 s, primer binding specific temperature for 30 s, 72 ℃ for 30 s, 35 cycles; the final extension product at 72 ℃ for 4 min.
Caveat
1. The quality of primer design of MSP is a key factor in the success or failure of amplification.The primer design of MSP is different from the common PCR primer design, the principle of MSP primer design is: after the template DNA is treated with sulfite, the cytosine at the 5'-end of the CpG site in the CpG island of the promoter region of the genes that have been methylated will remain unchanged, and the cytosine at the 5'-end of the CpG site in the CpG island of the genes that are not methylated will be converted into uracil, i.e., C-U. Sequence differences between methylated and unmethylated primers were designed by MethPrimer software for PCR amplification. Methylated and unmethylated primers were designed by MethPrimer software for PCR amplification. the primer sequence of MSP should contain at least one CpG site, preferably more than one CpG site, so as to ensure the specificity of the primers and to increase the detection rate of methylated bases in the DNA promoter. the primers of MSP should be designed according to the sequence of DNA after sodium bisulphite treatment. The primers of MSP must be designed according to the sequence of DNA after sodium bisulfite treatment, and at the same time, they should be compatible with the primer design principle of common PCR as far as possible. According to the requirement of MSP, at least two pairs of primers, i.e. methylated primers and unmethylated primers, should be synthesized for any DNA sequence for MSP amplification. In the unmethylated primer sequence for MSP, the forward primer does not contain guanine bases and the reverse primer does not contain cytosine bases. First-time MSP designers are advised to use literature primers for their studies. If you want to screen for new methylated oncogenes and cannot find the corresponding MSP primers in the literature, you can use the online MethPrimer software to design primers online (http://www.urogene.org/methprimer/indexl.html). The desired MSP primers can be found according to the software prompts. However, the difficulty encountered by MSP is that it is the promoter or part of the first exon sequence to be amplified, which has relatively high (C+G) content, and the difficulty of MSP amplification is increased. Therefore, it is better to use the primer software designed by the software, such as Primer 5.0, to test the amplification efficiency theoretically, and optimize the primers with the amplification efficiency of less than 30%, which can be adjusted before and after the core promoter region repeatedly. The method is to repeatedly adjust the methylation positive primers before and after the core promoter region to hit the starting point of amplification, in order to make the primer amplification efficiency increase, reduce the difficulty of amplification, so as to improve the PCR yield.
2、Sodium bisulfite modification of DNAThe purpose of DNA modification by sodium bisulfite is to convert unmethylated cytosine into uracil, while methylated 5-methylcytosine remains unchanged. The main factors affecting this process are the concentration of the modifying reagent, the temperature of the reaction, the pH of the reaction environment, and the reaction time. Problems in any one of them will lead to the failure of MSP amplification, as follows: ① The concentration of sodium bisulfite should be controlled at 3.0~3.9 mol/L, and the pH value should be adjusted to 5.0 with NaOH; ② The modification time should be 10~16 h. Too long a time for the modification will lead to the conversion of methylated cytosine into uracil and the increase of the destruction of the DNA template; too short a time will lead to the incomplete modification; ③ The modification time is too short; the modification time is too short; and the modification time is too short. Too long a modification time will lead to the conversion of methylated cytosine into uracil and increased destruction of the DNA template, while too short a time will lead to incomplete modification; (3) the reaction temperature should be controlled at 50~55 ℃ (if a domestic thermostatic water bath is used, it is recommended that the temperature be set at 53 ℃); (4) the amount of the DNA template should be controlled to be appropriate for ＜2 μg.
3、MSP reaction systemThe composition of the reaction system of MSP is the same as that of ordinary PCR, and the optimization of the reaction system is similar to that of ordinary PCR. The biggest difference between the optimization of the reaction system and that of ordinary PCR is that the DNA template is different. The modified template is single-stranded, and the purity and content of the DNA template of MSP should be detected after extraction, and its purity requires that the A260/A280 is between 1.8~2.0; the content should be detected accurately, otherwise, the DNA template is too much during the sulfite treatment, which leads to incomplete DNA processing and the failure of the MSP amplification; and if too little is added, then it will be a waste of reagents on the one hand, and on the other hand, the target fragment will be too little, which will lead to the failure of the MSP amplification. If too little DNA template was added, it would be a waste of reagents on the one hand, and on the other hand, it would lead to false-negative MSP because of too little target fragment.+MgThe concentration of Mg+ is usually 2.0~2.5 mmol/L, too low or too high is not favorable for amplification. In addition, the source of PCR buffer and Taq enzyme is also important, as general PCR buffer often leads to unstable results, and different sources of Taq enzyme may affect the reproducibility of results. We recommend using Takara's TaKaRa LATaq and GC buffers, which are rich in templates with complex secondary structures such as CG, for better results. Once a certain Taq enzyme is selected, do not change it easily to avoid the waste of time and cost caused by re-optimization of MSP.
4. Gel electrophoresis analysisMSP amplification results can be analyzed in three ways: (1) when the product is negative; (2) when the product shows multiple non-specific bands (including the target gene band); and (3) when the product is positive (target gene band only). The first two cases can be analyzed by gel electrophoresis, and the second case can be analyzed by gel electrophoresis. In the first two cases, the target gene bands can be obtained by adjusting the reaction temperature of the MSP while ensuring that the reaction system and primers are in good condition. The method is as follows: In the PCR reaction, the level of Tm is positively correlated with the (C+G) content. Due to the difference in sequence between methylated and unmethylated primers, PCR bias may occur in the amplification process.
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