What is sodium bisulfite used for?
A tank containing 25% sodium bisulfite at a water treatment plant in Sunnyvale, California. Sodium bisulfite is a common industrial reducing agent, as it readily reacts with dissolved oxygen: It is usually added to large piping systems to prevent oxidative corrosion.
What is bisulfite modification?
What is bisulfite modification? During DNA replication (cell division) methyl groups (DNA methylation) are resynthesized on the newly replicated strand by one of the enzymes called DNA Methyl Transferases (DNMT). Those enzymes are not present in standard PCR reaction.
What is a bisulfite test?
Bisulfite sequencing (also known as bisulphite sequencing) is the use of bisulfite treatment of DNA before routine sequencing to determine the pattern of methylation. DNA methylation was the first discovered epigenetic mark, and remains the most studied.
What is bisulfite treatment for DNA methylation?
Thus, bisulfite treatment introduces specific changes in the DNA sequence that depend on the methylation status of individual cytosine residues, yielding single-nucleotide resolution information about the methylation status of a segment of DNA.
What is the purpose of bisulfite conversion?
Sodium bisulfite conversion of genomic DNA is the most commonly used method for DNA methylation studies providing single nucleotide resolution. It enables to differentiate and detect unmethylated versus methylated cytosines.
How does bisulfite treatment of DNA make it possible to determine whether residues are methylated or non methylated?
After treatment with sodium bisulfite, unmethylated cytosine residues are converted to uracil whereas 5-methylcytosine (5mC) remains unaffected. After PCR amplification, uracil residues are converted to thymine. DNA methylation status can be determined by direct PCR sequencing or cloning sequencing.
What is bisulfite modification?
What is bisulfite DNA modification? Sodium bisulfite modification, also known as bisulfite conversion, is one of the most useful tools for analyzing methylation of cytosine residues at position 5. This method is based on treating DNA with sodium bisulfite in order to determine its methylation pattern.
What is the principle for bisulfite sequencing?
Bisulfite sequencing relies on the conversion of every single unmethylated cytosine residue to uracil. If conversion is incomplete, the subsequent analysis will incorrectly interpret the unconverted unmethylated cytosines as methylated cytosines, resulting in false positive results for methylation.
Is bisulfite converted DNA single stranded?
In bisulfite conversion, DNA is first denatured so that it is single stranded. The addition of sodium bisulfite will sulfonate unmethylated cytosines at a low pH, which then undergo spontaneous deamination into sulfonated uracil molecules, and subsequent desulfonation results in uracil molecules [28].
What is sodium bisulfite mapping?
Sodium bisulfite conversion of genomic DNA to differentiate and detect unmethylated versus methylated cytosines is the gold standard for DNA methylation analysis. It provides single nucleotide resolution map of 5-mC of the genome (1).
What happens during methylation?
DNA methylation is a biological process by which methyl groups are added to the DNA molecule. Methylation can change the activity of a DNA segment without changing the sequence. When located in a gene promoter, DNA methylation typically acts to repress gene transcription.
How long is bisulfite sequencing?
Following bisulfite treatment and purification, PCR amplification along with PCR product purification requires about 3 hours. Samples are then prepared for sequencing. If sending the samples to a sequencing core, time requirements may vary, but 3 days should be allotted for sequencing by a core facility.
What is pyrosequencing used for?
Pyrosequencing is used to reveal the genetic code of a section of DNA. It is also able to detect single nucleotide polymorphisms, insertion-deletions or other sequence variations, in addition to being able to quantify DNA methylation and allele frequency.
How does methylation PCR work?
Methylation-specific PCR (MS-PCR or MSP) is one of the most commonly used methods for gene/sequence-specific detection of DNA methylation. The DNA undergoes bisulfite conversion of cytosine to uracil and then the methylated sequences are selectively amplified with primers specific for methylation.
How much DNA is needed for bisulfite sequencing?
Commonly 10–20 samples are sufficient, but for some applications as many as 50 may be necessary.
How does DNA methylation happen?
DNA methylation is catalyzed by a family of DNA methyltransferases (Dnmts) that transfer a methyl group from S-adenyl methionine (SAM) to the fifth carbon of a cytosine residue to form 5mC (Figure 1).
What is bisulfite sequencing?
Bisulfite sequencing (also known as bisulphite sequencing) is the use of bisulfite treatment of DNA before routine sequencing to determine the pattern of methylation. DNA methylation was the first discovered epigenetic mark, and remains the most studied. In animals it predominantly involves the addition of a methyl group to the carbon-5 position ...
How to differentiate bisulfite from unconverted DNA?
A further method to differentiate converted from unconverted bisulfite-treated DNA is using high-resolution melting analysis (HRM), a quantitative PCR -based technique initially designed to distinguish SNPs. The PCR amplicons are analyzed directly by temperature ramping and resulting liberation of an intercalating fluorescent dye during melting. The degree of methylation, as represented by the C-to-T content in the amplicon, determines the rapidity of melting and consequent release of the dye. This method allows direct quantitation in a single-tube assay, but assesses methylation in the amplified region as a whole rather than at specific CpG sites .
What is pyrosequencing used for?
Pyrosequencing has also been used to analyze bisulfite-treated DNA without using methylation-specific PCR. Following PCR amplification of the region of interest, pyrosequencing is used to determine the bisulfite-converted sequence of specific CpG sites in the region.
Does bisulfite affect DNA?
Therefore, DNA that has been treated with bisulfite retains only methylated cytos ines.
Can bisulfite be used to desulfinate pyrimidine?
A potentially significant problem following bisulfite treatment is incomplete desulfonation of pyrimidine residues due to inadequate alkalization of the solution. This may inhibit some DNA polymerases, rendering subsequent PCR difficult. However, this situation can be avoided by monitoring the pH of the solution to ensure that desulfonation will be complete.
Is DNA bisulfite-converted?
DNA is bisulfite-converted, and bisulfite-specific primers are annealed to the sequence up to the base pair immediately before the CpG of interest. The primer is allowed to extend one base pair into the C (or T) using DNA polymerase terminating dideoxynucleotides, and the ratio of C to T is determined quantitatively.
Can bisulfite be used to identify 5-methylcytosine?
Therefore, bisulfite sequencing cannot discriminate between 5-methylcytosine and 5-hydroxymethylcytosine. This means that the output from bisulfite sequencing can no longer be defined as solely DNA methylation, as it is the composite of 5-methylcytosine and 5-hydroxymethylcytosine.
What is bisulfite DNA treatment?
Sodium bisulfite DNA treatment allows for discrimination between methylated and unmethylated cytosines. Bisulfite conversion was one of the first techniques developed for methylation analysis and is still considered the gold standard [21] because it allows for quantitative comparisons of methylation levels at single-base resolution. The main limitation of this assay is the inability to discriminate between hydroxymethylcytosine from methylcytosine.
What is the purpose of bisulfite conversion?
Bisulfite conversion of DNA is an essential first step for many gene-specific and epigenome-wide analyses. Since current sequencing methods cannot distinguish between methylated and unmethylated cytosines, different methylation-specific sequences must be created.
What is bisulfite sequencing?
Bisulfite genomic sequencing (BGS) generates the highest resolution map of the DNA methylation status of every single cytosine residue within a locus-of-interest, by the use of bisulfite conversion, PCR, cloning, and sequencing [21]. Bisulfite-treated DNA samples are amplified by PCR, and the PCR products are sequenced directly or sequenced after cloning procedures. Samples are first amplified by primers, which allow unbiased amplification of both methylated and unmethylated alleles. The amplicons are then cloned and sequenced. Sequencing of individually cloned products by Sanger Sequencing can reveal methylation of dozens of sites and allelic-specific methylation. Direct sequence analyses are performed to screen the major propensity of the DNA methylation status, whereas cloned sequence analyses are performed for the sake an accurate confirmation of methylation status, even down to single molecules. Similar to PMA, it is labor-intensive and hence is limited to small number of locus-of-interest and samples. Thus, for cancer screening, it is not an appropriate method.
Can bisulfite be detected by PCR?
Bisulfite-modified DNA can be detected by PCR. Combined bisulfite restriction analysis (COBRA) is a well-established bisulfite-based method developed in 1990s [135]. This technique is based on the fact that bisulfite treatment can lead to the creation of new endonuclease sites or to the elimination of pre-existing sites, so that some endonucleases are able to distinguish methylated from unmethylated sequences when digesting PCR products of bisulfite-treated DNA. COBRA is a semi-quantitative method since the level of methylation is linearly correlated with the relative proportions of digested and undigested products. Whereas, it can be affected by incomplete bisulfite conversion and/or partial digestion may affect the accurate quantification. COBRA method is low-throughput and relatively labor-intensive. And an improved protocol for COBRA, called Bio-COBRA, has been developed with a microfluidic platform for more high-throughput, accurate, and quantitative DNA methylation analysis [136].
Can bisulfite be used for 5mC?
Bisulfite conversion alone can no longer claim to be a gold standard in 5mC detection as it co-detects 5mC and 5hmC modifications, leaving a confounded signal. To address this limitation of bisulfite-only conversion, several methods have been developed to distinguish between the distinct cytosine modifications (with focus on 5mC and 5hmC). A recent review compares a selection of 5hmC specific profiling techniques (Skvortsova et al., 2017 ), in this article we focus on techniques compatible with 5hmC, 5fC and 5caC profiling ( Table 1 ).
What is sodium bisulfite?
Sodium bisulfite is a common industrial reducing agent, as it readily reacts with dissolved oxygen: It is usually added to large piping systems to prevent oxidative corrosion.
Is sodium bisulfite a compound?
Sodium bisulfite (or sodium bisulphite, sodium hydrogen sulfite) is a chemical mixture with the approximate chemical formula NaHSO 3. Sodium bisulfite in fact is not a real compound, but a mixture of salts that dissolve in water to give solutions composed of sodium and bisulfite ions.
What is sodium bisulfate used for?
Uses for Sodium Bisulfate. Sodium bisulfate is often used in toilet bowl cleaners and dishwasher cleaning products. It can also be found in a multitude of other industries. Commonly used to control pH, it is found in aquarium and swimming pool products.
Is sodium bisulfate a sulfate?
As noted in the U.S. Department of Health and Human Services' Household Products Database, sodium bisulfate is one of the many synonyms for sodium acid sulfate. It is a dry acid in crystal, granular, or powder form that is used as a pH adjuster, fungicide, herbicide, or microbiocide (a product that kills microbes) in a variety of industries, ...
Overview
Bisulfite sequencing (also known as bisulphite sequencing) is the use of bisulfite treatment of DNA before routine sequencing to determine the pattern of methylation. DNA methylation was the first discovered epigenetic mark, and remains the most studied. In animals it predominantly involves the addition of a methyl group to the carbon-5 position of cytosine residues of the dinucleotide C…
Methods
Bisulfite sequencing applies routine sequencing methods on bisulfite-treated genomic DNA to determine methylation status at CpG dinucleotides. Other non-sequencing strategies are also employed to interrogate the methylation at specific loci or at a genome-wide level. All strategies assume that bisulfite-induced conversion of unmethylated cytosines to uracil is complete, and this s…
Limitations
Bisulfite sequencing is used widely across mammalian genomes, however complications have arisen with the discovery of a new mammalian DNA modification 5-hydroxymethylcytosine. 5-Hydroxymethylcytosine converts to cytosine-5-methylsulfonate upon bisulfite treatment, which then reads as a C when sequenced. Therefore, bisulfite sequencing cannot discriminate between 5-methylcytosine and 5-hydroxymethylcytosine. This means that the output from bisulfite seque…
Applications: genome-wide methylation analysis
The advances in bisulfite sequencing have led to the possibility of applying them at a genome-wide scale, where, previously, global measure of DNA methylation was feasible only using other techniques, such as Restriction landmark genomic scanning. The mapping of the human epigenome is seen by many scientists as the logical follow-up to the completion of the Human Genome Project. This epigenomic information will be important in understanding how the functi…
Oxidative bisulfite sequencing
5-Methylcytosine and 5-hydroxymethylcytosine both read as a C in bisulfite sequencing. Oxidative bisulfite sequencing is a method to discriminate between 5-methylcytosine and 5-hydroxymethylcytosine at single base resolution. The method employs a specific (Tet-assisted) chemical oxidation of 5-hydroxymethylcytosine to 5-formylcytosine, which subsequently converts to uracil during bisulfite treatment. The only base that then reads as a C is 5‑methylcytosine, givi…
See also
• Reduced representation bisulfite sequencing
External links
• Bisulfite conversion protocol
• Human Epigenome Project (HEP) - Data — by the Sanger Institute
• The Epigenome Network of Excellence