Methylation PCR

Summary

Methylation-Specific PCR (MS-PCR) is a new method developed by Herman et al. (1996) based on the use of bisulfite treatment. This method is highly sensitive and is mainly used for qualitative studies. Methylation-specific PCR (MSPCR) is based on the transformation of cytosine to uracil from the parent's unmethylated sequences after treatment of DNA with sodium bisulfite, whereas the methylated sequences from the mother's side remain unchanged, and are subsequently amplified with highly specific primers.

Operation method

Methylation PCR

Materials and Instruments

Genomic DNA
Proteinase K RNAase Double-distilled water NaOH Hydroquinone
UV spectrophotometer Water bath Sodium bisulfite EP tube Aluminum foil Pipette gun

Move

I. Extraction of genomic DNA
This step focuses on the purity of the DNA, i.e. it is important to minimize or avoid contamination by RNA and protein. Therefore, proteinase K and RNAase are required to remove both during the extraction process.Details of using both:
1. Proteinase K may be prepared to 20 mg/ml using sterilized double-distilled water;
2. The RNAase has to be formulated without DNAase, i.e. reprocessed after purchasing commercially available RNAase and formulated to 10 mg/ml. otherwise the possible consequence is that not only is there no RNA, but even the DNA is digested. Both are stored at -20°C.
3. There are two methods for verifying the purity of extracted DNA:
(1) OD ratio calculated by UV spectrophotometer;
(2) 1%-1.5% agarose gel electrophoresis.
II. Genomic DNA modification by sodium bisulfite
The double distilled water (DDW) used was autoclaved if not otherwise noted.
1. Dilute approximately 2 ug DNA to 50 ul in a 1.5 ml EP tube using DDW;
2. Add 5.5 ul of freshly prepared 3 M NaOH;
3. 42°C water bath for 30 min;
Formulated during the water bath:
4. 10 mM hydroquinone (hydroxyquinone), add 30 ul to the above mixture after the water bath; (the solution turns pale yellow).
5. 3.6 M sodium bisulfite (Sigma, S9000), preparation: 1.88 g of sodium bisulfite was diluted with DDW and the solution titrated to pH 5.0 with 3 M NaOH to a final volume of 5 ml. Sodium bisulfite at this concentration is very difficult to dissolve, but it will dissolve slowly with the addition of NaOH, so patience is necessary. pH must be exactly 5.0. 520 ul of the solution was added to the above water bath. to the solution after the above water bath.
6. Wrap the EP tube in aluminum foil, protect from light and mix the solution by gentle inversion.
7. Add 200 ul of paraffin oil to prevent water evaporation and limit oxidation.
8. Water bath at 50°C for 16 h, protected from light.
Generally, this step is started at 4 pm, and can be completed in less than 5 pm if you are skillful, and the water bath is 16 h just until after 8 am the next day, which is a very suitable time.
This step detail:
(1) The amount of genomic DNA does not need to be very precise, and it is better to have more than less, as it will be lost in later purification and recovery steps, and the method can be modified up to 4 ug.
(2) All reagents must be freshly prepared, so the technique of dispensing the liquid must be overly technical, both fast and precise.
(3) Sodium bisulfite solution is strongly acidic, be sure to use alkali to modulate the PH 5.0, otherwise the PH is not suitable will affect the subsequent purification and absorption.
(4) Water baths are preferable for up to 16 hours, although they can be as short as 8 hours, but the latter modification will be incomplete.
III. Purification and recovery of modified DNA
EP tubes are autoclaved if not otherwise specified.
1. Insert the tip of the pipette under the layer of paraffin oil, press gently to expel a small portion of the paraffin oil, and then aspirate the mixture into a clean 1.5 ml EP tube.
2. The Promega Wizard Cleanup DNA purification and recovery system (Promega, A7280) was used as follows.
(1) Preheat DDW in a 70°C water bath; prepare 80% isopropyl alcohol;
(2) Add 1 ml of Promega's Wizard DNA Clean-up resin and mix with gentle inversion to fully bind the DNA to the resin;
(3) Since the kit is equipped only with a syringe without a spigot, it is easy to use if there is a vacuum-negative-pressure suction device; if not, it is necessary to provide a 3 ml~5 ml syringe by oneself. Tightly connect the syringe barrel to the recovery column provided in the kit, transfer the above mixture to the barrel with a pipette, and place an EP tube of 2 ml or more under the column to receive the waste liquid. Add the needle plug, gently apply pressure and squeeze out the liquid, at this time, white resin deposits can be seen inside the column.
(4) Separate the syringe from the column and pull out the pin, then connect the syringe to the column, add 2 ml of 80% isopropanol to the syringe, insert the pin and gently pressurize to squeeze out the isopropanol. This is the washing step.
(5) Separate the syringe from the column, place the column on a clean 1.5 ml clean EP tube, and centrifuge at 12,000 rpm for 2 min to remove residual isopropanol content and dry the resin. At this point, the modified DNA is in the resin-bound state.
(6) Remove the column and place it on another clean 1.5 ml EP tube, pipette in 50 ul of pre-warmed DDW and leave for 5 min at room temperature.
(7) Centrifuge at 12 000 rpm for 20 s. This is the elution step, and the liquid in the EP tube at this time is the eluted modified DNA solution, with a final volume of 50 ul.
3. Add 5.5 ul of freshly prepared 3 M NaOH and leave for 15 min at room temperature.
4. 33 ul 10 M ammonium acetate was added to neutralize the NaOH and bring the solution pH to about 7.0.
5. Add 4 ul 10 mg/ml of glycogen, which is used as a precipitation indicator because it produces a precipitate when mixed with ethanol, making it easier to identify the location of the recovered material later after centrifugation to prevent it from being sucked out when the residual ethanol is aspirated. In fact, it's hard to say just how much adding these glycogen will help. However, there are domestic glycogen for sale in small packages and cheap, so buy it and use it, counting on strict adherence to the steps in the literature.
6. Add 270 ul of ice-anhydrous ethanol, place at -20 degrees Celsius, and allow to settle overnight. Some have suggested as little as 2 hours for precipitation, but I think longer may be better. And by the time you do this step, it will usually be noon, or afternoon if there are a lot of samples, so you may want to leave it overnight to ease your schedule and do some other tests along the way. If you want to do it the same day, no problem, but I think it's better to let it settle longer, at least 6 hours.
7. 4 degrees Celsius, centrifuge at 12 000 rpm for 30 min, pour off the supernatant and collect the precipitate. It is not necessary to aspirate.
8. Add 500 ul of 70% ethanol, do not blow up the precipitate, just add the ethanol. Gently tilt the EP tube, spin it around and centrifuge again at 4 degrees 12 000 rpm for 5 min. after centrifugation pour off the supernatant and add the same amount of ethanol and do the same again. This is the washing step, 2 times in total.
9. Pour off the supernatant, and after brief centrifugation at room temperature, remove the attached ethanol to the bottom of the EP tube, pipette the residual liquid carefully, and dry it for 5 min at room temperature, or when the precipitate changes from opaque to semi-transparent or transparent, add 20 ul~30 ul of DDW to dissolve the precipitate. At this point, the purification and recovery of modified DNA has been completed, and the result is a modified DNA solution that can be used for further experiments thereafter.
10. Store the DNA solution at -20°C.
This step details:
(1) When using the syringe, the force must be even and light; if violence is used, the membrane inside the column will be squeezed and become useless.
(2) Ammonium acetate and glycogen do not need to be freshly prepared, glycogen is stored at -20 degrees after preparation, and ammonium acetate can be stored at room temperature because ammonium acetate at this concentration is very difficult to dissolve, and once it is placed at 4 degrees, a lot of solute will be precipitated when it is taken out of use.
(3) Isopropyl alcohol and 70% ethanol do not need to be freshly prepared, but it is easy to prepare them on site if the amount is large.
The key to this step is in the combination of resin and DNA, which re-emphasizes the importance of the second part of the pH value of sodium sulfite. Because the resin and DNA binding needs to have a proper pH, if the previous step is not good, the resin can not be well combined with the DNA in this step, will bring disastrous consequences, that is, the DNA with the liquid was extruded, the elution of the actual no longer any DNA.
IV. Modified DNA for PCR
There's no suspense in this step either. I'm going to focus on a couple of the tougher issues here:
1. Primer problem: I feel that it is quite difficult to design your own primers, I have designed a few pairs of primers and tested them, but they failed. If you have enough time and you are working on a relatively new gene, there is not much literature, you can design your own primers without any problem. If not, it is better to refer to the literature. First of all, check the literature with high SCI scores, then the literature of famous laboratories, and if there are domestic ones, even better, you can contact them directly for advice. After checking the sequence, make sure to compare it with the sequence in Genbank, to prevent the difference of individual bases caused by printing errors. Then search on Google to see whether there are many people using it and whether the system conditions are the same. If there are a lot of people using it and the system conditions are the same, it means that it is more reproducible. I did the same thing, and it went well.
2. Taq enzyme issue: there is literature on using high fidelity Gold Taq (Platinum), but I feel that a normal hot start enzyme is fine as long as the system is correct, denaturation annealing and other conditions are right. I started using Takara's LA Taq, which works well and comes with 10x LA buffer containing mg2+. Sometimes I run out of it, so I can use Takara's normal Taq enzyme for a while. You can choose according to your own situation. First time authors should use a better enzyme.
3. PCR conditions: denaturation is usually 95 degrees for 3 min, the rest I feel is still according to the literature, annealing can be tried in a small range according to the annealing temperature of your primers. Generally, there is not much difference from the literature. It's just a matter of amplification fragment specificity. It is recommended to follow the literature.
4. It is best to choose imported EP tubes for PCR, with thin walls and uniform thickness, which can ensure that rapid changes in temperature can be transferred to the reaction solution inside the tube in time, so that the system really operates at the set temperature.
5. PCR instrument: If you have made it on a certain instrument, it is better to continue with that instrument. Different instruments have different "temperaments", but the EP tube must be tightly fitted to the jack inside the instrument, leaving gaps that, in my opinion, will affect the temperature transfer.
V. Gel recovery of PCR products
This step is relatively simple, you can buy a gel PCR product recovery kit, the domestic is very good and reasonably priced, such as TIANGEN products (used). Cut down the gel according to the instructions can be operated.
A few details:
1. PCR products are subjected to agarose gel electrophoresis using freshly prepared electrophoresis solution. Gel concentrations of 1%-2% are acceptable.
2. Observe the position of the bands under 300nm UV light when recovering DNA from the gel, and cut the gel where the target fragment is located, as small as possible to ensure specificity.
3. The duration of UV exposure should not be too long, otherwise there will be damage to DNA.
4. Recovered DNA is stable for several months when stored at -20 degrees Celsius if not used immediately.
VI. Ligation and transformation of PCR products with T vector, blue and white spot screening
1. Ligation of T vector (Promega's kit was used in this experiment)15 ul systemT-easy 1 ulLigase 1 ul2×buffer 7.5 ulDNA 5.5 ul4 degrees, overnight.
2. Transformation of conjugation products
(1) Remove the sensory bacteria from the -70 degree refrigerator, melt and place on ice;
(2) Add all 15ul of the ligation product to the ice for 30 minutes;
(3) 42 degrees, 90 seconds;
(4) 2 minutes on ice;
(5) 800 ul LB medium;
(6) 280 rpm, 37 degrees Celsius, shaker for 45 minutes (place the tube horizontally and shake to ensure that the liquid is shaken well);
(7) 8,000 rpm for 1 min; remove supernatant in an ultra-clean bench and retain 100-150 ul;
(8) Coated plates: incubator at 37 degrees overnight; (plates are solid LB medium containing ampicillin)Coat first: X-gar 35 ulIPTG 25 ulPost-coating: suspensionAfter overnight many blue or white spots are visible growing on the plate, take the white spots, especially around the blue spots, where the self-association rate is low.
3. Remove the white spot and plant it on a new plate.
New plate: apply first: X-gar 35 ul
IPTG 25 ul
Then mark the bottom of the plate with a partition. Depending on the need, it is usually not a problem to make 50 clones per plate.
The needle picks the white spot and makes 2 strokes in the corresponding area on the plate.

Incubate overnight at 37 degrees.4. Contact a sequencing company to send sequencing. Usually a clone is 35~45 dollars.
The details of this section:
(1) Apply the board uniformly to ensure that Xgar and IPTG are evenly distributed on the board surface;
(2) Don't let the blue and white spots grow too full or you'll be prone to picking 2 at once when selecting clones.

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