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Bisulfite sequencing is a sensitive method of analyzing DNA methylation, which can be used to determine active regions of a genome and identify gene-rich areas. However, it requires high salt concentrations and may result in false positive calls. It cannot be used on living people but is useful for analyzing clinical samples.
Bisulfite sequencing is a method in which different regions of DNA are analyzed using methylation. Methylation is the process of adding a specific molecule, called a methyl group, to a nucleotide, in this case usually a cytosine. Inactive nucleotides are often methylated, so this method can be used for a variety of purposes, from determining the active regions of a genome to identifying gene-rich regions. In bisulfite sequencing, methylated cytosines are unaffected by the sequencing process, while unmethylated cytosines are converted to uracil, a nucleotide not usually found in the genetic material, deoxyribonucleic acid (DNA).
This method is very sensitive to changes in methylation, so small changes in binding can give researchers specific information about particular nucleotides. Sodium bisulfite converts cytosine to uracil, but the conversion occurs in an environment where methylated cytosine will not undergo this change. When bisulfite sequencing is complete, the original DNA has been converted into a significantly different molecule. Cytosines will be markedly reduced or potentially absent. If a cytosine is still found in this converted molecule, it represents a naturally methylated cytosine in the genome under study.
Like all experimental protocols, bisulfite sequencing has disadvantages. Its most significant drawback is that it requires a very high salt concentration to function properly. The salt encourages the annealing of single-stranded DNA into its more natural double helix, and sodium bisulfite cannot always reach the cytosines when they are part of double-stranded DNA. If the salt concentration is too high, a number of cytosines may not be converted to uracil, resulting in false identification of methylated cytosines within a genome. Denaturing agents may be needed to minimize the number of false positive calls.
Large amounts of genomic data are not required for bisulfite sequencing, so the method has a useful application in the analysis of clinical samples. The original source of the nucleic acid does not matter, but the source must be DNA. In theory, ribonucleic acid (RNA) could be sequenced using this method, since most RNA is single-stranded and would not be as susceptible to false positives due to blocked nucleotides. When applied, however, bisulfite sequencing is not useful for RNA, because RNA naturally contains uracil. Without some sort of external labeling or addition to the protocol, the converted cytosines would be indistinguishable from natural uracil.
When undertaking any type of sequencing methodology, accuracy and precision are essential. Sensitive methods such as bisulfite sequencing offer a reliable means of sequence analysis, which in turn enables gene analysis and identification of drug and therapeutic targets. While this method cannot be used on living people, it can still be of great help with only the smallest of tissue samples to work with.
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