Detection of methylation by sequencing after bisulfite modification

Summary

Bisulfite-modified sequencing can be used primarily to detect methylation.

Operation method

DNA methylation

Principle

Heavy sulfite deamidates the unmethylated cytosine in the DNA into uracil, while the methylated cytosine remains unchanged, and all of the uracil is converted into thymine when the desired fragment is amplified by PCR (primers are designed to avoid having CpG as much as possible so that they will not be affected by the methylation factor). Finally, the PCR product was sequenced and compared with the untreated sequence to determine whether the methylation status of the CpG site.

Materials and Instruments

DNA
NaOH Phenol (hydroquinone) Sodium bisulfite Paraffin oil
Centrifuge tube Constant temperature water bath Aluminum foil

Move

1. dilute about 2ug of DNA to 50ul in a 1.5ml EP tube using DDW;2. Add 5.5ul of freshly prepared 3M 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 water bath; (solution turns pale yellow)5. 3.6M sodium bisulfite (Sigma, S9000), preparation: 1.88g of sodium bisulfite was diluted using DDW and the solution titrated with 3M NaOH to a final volume of 5 ml of PH 5.0. Such a large concentration of sodium bisulfite is very difficult to dissolve, but it will dissolve slowly with the addition of NaOH, so patience is needed. the PH must be exactly 5.0. 520ul was added to the solution after the above water bath.6. Wrap the outside of the EP tube with aluminum foil, protect from light, and mix the solution by gently inverting it.7. Add 200 ul of paraffin oil to prevent water evaporation and limit oxidation.8.50°C water bath with light protection for 16h.

Caveat

Avoid contamination with chemicals or exogenous DNA.

Common Problems

Methylation is the current research hotspot, on the little work I have done and one of the little insights, to share with you. I hope it can be helpful to you.

Part I Genomic DNA extraction.

There is no suspense in this step, you can buy DNA extraction kits for cells or tissues, and if the laboratory conditions are ripe, it is perfectly possible to extract the DNA with your own reagents. the DNA is relatively stable, as long as you do not use violence in the operation, the proposed genomic DNA should be intact.

This step focuses on the purity of DNA, that is, it is important to reduce or avoid the contamination of RNA and protein. Therefore, proteinase K and RNAase are used to remove both during the extraction process.

Details of using both:

1: Proteinase K can be prepared to 20mg/ml using sterilized double distilled water;

2: RNAase must be prepared without DNAase, i.e., after purchasing commercially available RNAase, reprocess it to 10 mg/ml, otherwise not only is there no RNA, but also the DNA is digested as a possible consequence. Both were stored at -20 degrees.

There are two ways to verify the purity of the extracted DNA:

1: UV spectrophotometer to calculate the OD ratio;

2: 1%-1.5% agarose gel electrophoresis.

I tend to prefer the second method, which can completely clarify the purity of the proposed genomic DNA and estimate its concentration based on the up-sampling volume of Marker for the next step of modification.

Part II Genomic DNA modification by sodium bisulfite

The double distilled water (DDW) used was autoclaved if not otherwise noted.

1: Dilute about 2ug of DNA in a 1.5ml EP tube to 50ul using DDW;

2: Add 5.5ul of freshly prepared 3M NaOH;

3: Water bath at 42°C for 30min;

Prepared during water bath:

4: 10mM hydroquinone (hydroquinone), add 30ul to the above mixture after the water bath; (the solution turns light yellow)

5: 3.6M sodium bisulfite (Sigma, S9000), preparation: 1.88g of sodium bisulfite was diluted with DDW and the solution was titrated with 3M NaOH to a final volume of 5 ml of PH 5.0. Such a large concentration of sodium bisulfite is very difficult to dissolve, but it will dissolve slowly with the addition of NaOH, so patience is required. the PH must be exactly 5.0. 520 ul was added to the above post-water bath solution. the above solution after the water bath.

6: Wrap the EP tube with aluminum foil, protect it from light, and mix the solution by gently inverting it.

7: Add 200 ul of paraffin oil to prevent water evaporation and limit oxidation.

8: Water bath at 50°C for 16h, protected from light.

Generally, this step is started at 4pm, but can be finished in less than 5pm if you are skillful, and the water bath for 16h is just until the next day after 8am, which is a very suitable time.

This step details:

1: The amount of genomic DNA does not need to be very precise, rather more than less, because there will be lost in the later purification and recovery steps, and this method can be modified up to 4ug.

2: All reagents must be freshly prepared, so the technique of liquid preparation should be over the top, both fast and precise.

3: Sodium bisulfite solution is strongly acidic, be sure to use alkali to modulate PH 5.0, otherwise the PH is not appropriate will affect the subsequent purification and absorption.

4: The water bath is best up to 16 hours, although it can be as short as 8 hours, but the latter modification will have incomplete.

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