Bisulfite Sequencing - An Overview

Introduction:

DNA methylation is a type of epigenetic mutation (alteration in the chemical structure of DNA and not in the DNA (deoxyribonucleic acid) sequence, which can change gene expression; these changes occur due to exposure to chemicals, drugs, and environmental factors such as tobacco, smoke, diet, cadmium metal, and Epstein-Barr virus) where a methyl group is added to 5th carbon of cytosine in CpG dinucleotides with the help of DNA methyltransferases. This methyl group is obtained from S adenosine methionine (SAM), these levels are increased by the intake of vitamins like folic acid, vitamin B6, and vitamin B12. DNA methylation can be inherited from parents. These can affect tissue development, cellular proliferation, chromosome stability, and cell differentiation, which can cause human diseases such as cancer. Bisulfite sequencing can help to find the exact place of 5-methylcytosine (m5C) on single-stranded DNA. When DNA is treated with bisulfite, it converts cytosine to uracil, and methylated cytosine is left unaffected.

What Is Bisulfite Sequencing?

Bisulfite sequencing was developed by Frommer and colleagues. It detects DNA methylation and helps in identifying 5-methylcytosine at a single base-pair resolution. When the DNA is treated with sodium bisulfite, the cytosines are converted into uracil residues known as thiamin, and the methylated cytosine is left unaffected. This helps in identifying methylated and non-methylated cytosines. This status is determined by using direct PCR (polymerized chain reaction) product sequencing and sub-cloning sequencing. This sequencing provides a quantitative, qualitative, efficient approach and also helps in the detection of DNA methylation patterns in double-stranded DNA by denaturing (converting double-stranded DNA into single-stranded) the DNA.

What Is The Method Of Bisulfite Sequencing?

The steps in bisulfite sequencing include five steps which are denaturation of DNA, incubation with bisulfite, removing the bisulfite by desalting, desulfonation of sulfonyl uracil, and removing the desulfurized solution. About two micrograms of genomic DNA are used for this method. The steps involve;

• Genomic DNA is extracted from cultured cells, animal tissues, cultured bacteria, and paraffin-embedded tissue sections, by using DNA extraction kits and it is dissolved in 18 microlites of deionized water.

• The extracted DNA is pre-denatured in boiling water for twenty minutes.

• The DNA is denatured by using two microliters of 3 M freshly made sodium hydroxide and 380 microliters of 5 M sodium bisulfite solution.

• 500 microliter of heavy mineral oil is added along with the above-mentioned solution to decrease evaporation and is incubated in the dark at 50 degrees Celsius for 12 to 16 hours.

• Bisulfite treated-DNA is purified using a wizard DNA clean–up kit.

• The bisulfite-modified DNA is eluted (extracting the DNA by washing in 50 microliters of deionized water) and 11 microliters of 3 M NaOH is added and incubated at 37-degree Celsius for 15 minutes for desulfonation of DNA.

• Precipitation of DNA is done by adding 166 microliters of 5 M ammonium acetate, 200 microlite isopropanol, and 750 microlites of absolute ethanol, which is done at −20 degree Celsius for two to four hours.

• The DNA is centrifuged for ten minutes.

• The DNA is then washed with 200 microliters of 70 percent ethanol and a centrifuge is carried on.

• The ethanol is removed and DNA is dried for ten minutes at room temperature and the DNA is resuspended in 10 to 20 microliter of deionized water.

• The amplification or sub-cloning procedures include :

  • Bisulfite PCR amplification is done and the results are verified using gel-based electrophoresis. Successful PCR amplification is shown as a single and bright band.

  • Direct PCR sequencing is done by purifying the PCR products using available kits and sequencing is done directly. It can limit its application by not reading the entire target region.

  • Cloning sequencing is done to observe the methylation pattern. The cloning is done in JM109 cells by using a pGEM-T Easy vector, which is done on agar plates containing ampicillin or X-gal, or IPTG. It can provide molecular basis information.

• After amplification, sequencing analysis of DNA methylation is done. To obtain high accuracy, many clones should be sequenced.

• The sequence of methylation can be interpreted by comparing it with the original sequence.

• Cytosine converted to thymine is called T peak and unmethylated cytosines are called C peak. Total methylation occurs if only one peak is present, and partial methylation occurs when both peaks are present, which is incomplete bisulfite conversion.

What Are the Types of Bisulfite Sequencing?

The different types of bisulfite sequencing are:

• Whole Genome Bisulfite Next-Generation Sequencing: This type includes detection of DNA methylation in an entire genome which is called as methylome. This study requires large number of DNA.

• Reduced Representation Bisulfite Sequencing: This type is much simpler than whole genome bisulfite next-generation sequencing and requires less amount of DNA. In this, the GC-rich parts of DNA strand is digested by an enzyme that connects with CpG dinucleotides. Since DNA methylation are present on CpG dinucleotides, this type is useful.

• Targeted Bisulfite Sequencing: This type includes recognizing the methylation status in a specific region of DNA, rather than involving entire genome.

What Are The Advantages Of Bisulfite Sequencing?

The advantages of bisulfite sequencing are:

• To detect methylation patterns.

• Bisulfite sequencing uses single-base pair resolution.

• It is used in stem cell (undifferentiated cells that can become any type of cell) applications.

• It is also used in the early detection of diseases.

• It is used in forensic sciences.

• Cost-effective sequencing method.

• This sequencing provides a quantitative, qualitative, and efficient approach.

What Are The Limitations Of Bisulfite Sequencing?

The limitations of bisulfite sequencing are:

• Bisulfite sequencing cannot differentiate between 5-methylcytosine and 5-hydroxymethylcytosine.

• Bisulfite sequencing accuracy depends on complete conversion; incomplete conversion can result in interpreting non-methylated cytosines as methylated cytosines, which can give wrong results.

• Bisulfite can react with only single-stranded DNA, therefore denaturation of DNA should be done accurately.

• Factors such as high temperatures stretched incubation time, and high concentrations of bisulfite can lead to the degradation of DNA.

Conclusion:

Bisulfite genomic sequencing is a great method to detect DNA methylation. It is an efficient tool for studying epigenetic mutations. It has its own advantages such as detecting methylation patterns, early detection of diseases, stem cell applications, and providing quantitative, qualitative, and efficient approaches; and limitations such as cannot differentiate between 5-methylcytosine and 5-hydroxymethylcytosine, and can react with only single-stranded DNA.