Validation of DNA Methylation by Bisulfite Pyrosequencing

Validation of DNA Methylation by Bisulfite Pyrosequencing

Simran Kaur^1*, Amandeep Singh² and Rahul Kumar³

¹Ph.D. Scholar, Department of Animal Genetics and Breeding, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences (LUVAS), Hisar-125004, Haryana, India. Email: sudansimran321@gmail.com. Contact: +91-9086076546.

²Assistant Professor, Department of Veterinary Anatomy, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences (LUVAS), Hisar-125004, Haryana, India. Email: amanatomy287@gmail.com. Contact: +91-7082227569.

³Assistant Professor of Zoology, College of Agricultural Bawal-123501, Rewari, CCS Haryana Agricultural University, Hisar, Haryana, India. Email: rahulrohila01@gmail.com. Contact: +91-7404133861

*Corresponding author email: sudansimran321@gmail.com

ABSTRACT

Gene expression as well as emergence of several ailments is regulated by methylation of DNA. High throughput techniques like bisulfite conversion method, restriction enzyme and affinity enrichment method are employed for identifying methylation of CpGs in the genome. Bisulfite conversion is the most extensively used procedure for delineating methylated DNA from the DNA that is unmethylated. Pyrosequencing, which is a non-electrophoretic synthesis technique is commonly regarded as a promising approach to unravel methylation in DNA. The bisulfite method can assess several CpG methylation sites in one reaction, is convenient, has excellent reproducibility, and is very affordable. The majority of illnesses viz., neurological, cardiovascular, cancer have been linked to abnormal methylation. In order to comprehend the underlying biological process of these disorders, analysis of DNA methylation patterns is essential.

Keywords: DNA methylation, bisulfite conversion, pyrosequencing, CpGs

INTRODUCTION

CpG dinucleotide methylation is an epigenetic phenomenon which involves addition of methyl group (CH₃) to the carbon of cytosine at 5^th position particularly at cytosine-guanine dinucleotides known as CpG islands. DNA methyltransferases are the enzymes that are known to carry out the process of DNA methylation. High throughput techniques like bisulfite conversion method, restriction enzyme and affinity enrichment method are employed for identifying methylation of CpGs in the genome. Bisulfite pyrosequencing-based methylation detection method is a straightforward, highly reproducible, sensitive, a significant tool for biomarker identification and clinical diagnosis (Florea, 2016). Because of the consistency and accuracy of pyrosequencing, measurement variance is minimal, enhancing the chance of early identification of modest changes in methylation levels. Low measurement variation produced by consistency and precision of pyrosequencing increases the chance of spotting early methylation level changes that might become noticeable in response to therapy. For instance, the LINE-1 assay’s great repeatability is crucial for identifying the very little daily changes in methylation levels linked to hypomethylation.  This enables the identification of patterns that differ between healthy and sick tissue, such as those in tumor suppressor genes, as well as the assessment of total methylation changes in retaliation to pharmacological interventions. (Poulin et al., 2018). Pyrosequencing approach has opened up new avenues for sequence-based DNA analysis and can be utilized as a tool for studying genetic diversity.

Bisulfite conversion

Treatment by sodium bisulfite brings out deamination of nonmethylated cytosines into uracil while methylated cytosines are left unaltered in CpG dinucleotides (Pajares et al., 2021).

Principle of Bisulfite Pyrosequencing

Pyrosequencing, a non-electrophoretic synthesis method, provides real-time sequence information and is often used in genetic and epigenetic research and is widely recognized as a promising method for determining DNA methylation. It was first reported by Nyrén et al. in 1993.  The method changed the strategy to fix fragments of DNA on a fabricated microarray to enable massive parallel sequencing, and as the process developed and refined, it became the first commercially available, successful next-generation sequencing technology. But now that this technology for next-generation sequencing has been discontinued, pyrosequencing is currently mostly used for one-off research to assess regional methylation at a small number of CpG sites (Kumar et al., 2020).

The technique entails detecting inorganic pyrophosphate (PPi) produced during DNA synthesis. The reaction mixture is gradually supplemented with four deoxynucleotide triphosphates (dNTPs) in a predefined order. The ATP (adenosine triphosphate) sulfurylase (Saccharomyces cerevisiae) converts the released PPi to ATP in the presence of adenosine, which gives luciferase (Photinus pyralis) the energy it needs to oxidize luciferin and create light. The amount of pyrophosphate released controls the amount of light emitted (=560 nm), which is comparable to the number of nucleotides incorporated in the reaction (Harrington et al., 2013). The quantity of light detected by a charge-coupled device (CCD) camera corresponds to the amount of ATP released (about 6*10¹¹ ATP molecules are produced by one pmole of DNA, and these molecules yield 6*10⁹ photons). The dNTP and ATP that remain unincorporated are degraded and eliminated by apyrase (Solanum tuberosum) enzyme in order to make room for the subsequent nucleotide to be dispensed. The sequence information is described by peak presence or absence and peak heights depicted by a pyrogram (Ghemrawi et al., 2023). The methylation percentage is calculated by dividing the height of a cytosine peak (methylated signal) by the total of the heights of cytosine and thymine peaks (unmethylated and methylated signal) (Pajares et al., 2021).

PPi + APS ➞ ATP + Sulfate (catalyzed by ATP-sulfurylase)

luciferase

Lucifer               Oxyluciferin + CO₂ +

Advantages

1. The bisulfite approach is straightforward, extremely reproducible, has an outstanding quality/price ratio, and is capable of assessing several methylation positions at CpG in similar reaction.
2. The outcome of result is very standardized.
3. It permits the analysis of heterogeneous samples when a gene of interest with differential methylation is only present in a small group of cells.

Disadvantages

1. The inability to discriminate between 5mC and 5hmC alterations is the fundamental drawback of the bisulfite conversion.
2. During the process, single-stranded DNA may re-anneal, avoiding full cytosine conversion.
3. If the conversion is inadequate, unconverted cytosines will be misidentified as methylated cytosines, leading to misleading findings.
4. The number of CpG sites that may be evaluated is limited by the low number of base pairs analyzed per amplicon (about 50–60 base pairs).

CONCLUSION

Gaining a complete grasp of DNA methylation function in illnesses and developmental pathways has been made feasible by the quick development of novel techniques. Furthermore, DNA sequences with differential methylation may act as diagnostic, prognostic, and predictive markers in instances of cancer, autoimmune diseases, metabolic disorders, and neurological disorders. Therefore, by weighing the advantages and disadvantages of each approach, the optimum technique for methylation analysis of genome may be selected.

BIBLIOGRAPHY

1. Florea, A.M. (2016). Pyrosequencing and its application in epigenetic clinical diagnostics. In Epigenetic Biomarkers and Diagnostics, 175-194, Academic Press.
2. Ghemrawi, M., Tejero, N.F., Duncan, G. and McCord, B. (2023). Pyrosequencing: Current forensic methodology and future applications-A review. Electrophoresis, 44(1-2), pp.298-312.
3. Harrington, C.T., Lin, E.I., Olson, M.T. and Eshleman, J.R. (2013). Fundamentals of pyrosequencing. Archives of Pathology and Laboratory Medicine, 137(9), 1296-1303.
4. Kumar, A., Dalan, E. and Carless, M.A. (2020). Analysis of DNA methylation using pyrosequencing. In Epigenetics methods, 37-62, Academic Press.
5. Pajares, M.J., Palanca-Ballester, C., Urtasun, R., Alemany-Cosme, E., Lahoz, A. and Sandoval, J. (2021). Methods for analysis of specific DNA methylation status. Methods, 187, 3-12.
6. Poulin, M., Zhou, J.Y., Yan, L. and Shioda, T. (2018). Pyrosequencing methylation analysis. Cancer Epigenetics for Precision Medicine: Methods and Protocols, 283-296.

Please follow and like us: