What does bisulfite do to DNA?

Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected. Therefore, DNA that has been treated with bisulfite retains only methylated cytosines.

What is bisulfite PCR?

Bisulfite conversion involves the deamination of unmodified cytosines to uracil, leaving the modified bases 5-mC and 5-hmC. The uracils are amplified in subsequent PCR reaction as thymines, whereas 5-mC or 5-hmC residues get amplified as cytosines.

Is bisulfite converted DNA single stranded?

Uracil is then converted to thymine upon PCR amplification. Note that bisulfite-converted DNA is a single-stranded DNA because the two strands of DNA are no longer complementary after conversion.

What is bisulfite pyrosequencing?

Bisulfite pyrosequencing is a sequencing-by-synthesis method used to quantitatively determine the methylation of individual CG cytosines from PCR amplicons of a region up to 115 bases in length.

How much DNA is needed for bisulfite sequencing?

Up to 2 µg genomic DNA (or 1 ng purified fragment) may be used for BGS. Although 90% or more of DNA is lost during bisulfite treatment, using too much DNA may lead to incomplete deamination, due to re-annealing of complementary sequences.

Is bisulfite sequencing expensive?

Whole-genome bisulfite sequencing is currently the most expensive method of global methylation assay, but it provides many advantages while overcoming challenges present in pyrosequencing. With current multiplexing technology, only 24 samples can be combined into a single lane of WGBS sequencing.

Why does DNA methylation occur?

DNA methylation is an epigenetic mechanism that occurs by the addition of a methyl (CH3) group to DNA, thereby often modifying the function of the genes and affecting gene expression. When a CpG island in the promoter region of a gene is methylated, expression of the gene is repressed (it is turned off).

Who invented bisulfite sequencing?

Frommer et al
This method was first introduced by Frommer et al and it is based on the finding that the amination reactions of cytosine and 5-methylcytosine (5mC) proceed with very different consequences after the treatment of sodium bisulfite (6).

How do you confirm bisulfite conversion?

Following bisulfite conversion the unmethylated cytosines are converted into uracil while the methylated cytosines remain as cytosine. The methylation profile of the DNA can then be determined by PCR amplification followed by DNA sequencing, where the uracils will be detected as thymine.

What does bisulfite sequencing tell you?

Bisulfite sequencing is mainly used to detect DNA methylation patterns. As DNA methylation patterns are erased during PCR (polymerase chain reaction) amplification, current sequencing, and microarray technologies cannot distinguish between methylated and unmethylated cytosines.

Is DNA methylation reversible?

The pattern of DNA methylation plays an important role in regulating different genome functions. Thus, contrary to the commonly accepted model, DNA methylation is a reversible signal, similar to other physiological biochemical modifications.

How can DNA methylation be reduced?

Most of the existing research suggests that DNA methylation relies at least in part on folate, vitamin B-12, vitamin B-6, and choline, in addition to other vitamins and minerals. Increasing your intake of these nutrients may help to support DNA methylation, preventing certain genes from being expressed.

What is sodium bisulfite used for in DNA analysis?

Sodium bisulfite is used in the analysis of the methylation status of cytosines in DNA . In this technique, sodium bisulfite deaminates cytosine into uracil, but does not affect 5-methylcytosine, a methylated form of cytosine with a methyl group attached to carbon 5.

How is the genomic DNA amplified after bisulfite conversion?

Following bisulfite conversion, the genomic DNA is amplified with PCR that does not discriminate between methylated and non-methylated sequences. The numerous methods available are then used to make the discrimination based on the changes within the amplicon as a result of bisulfite conversion.

Which is bisulfite genomic sequencing protocol do you use?

The bisulfite genomic sequencing (BGS) proto- col has gained worldwide popularity as the me- thod of choice for analyzing DNA methylation. It is this popular because it is a powerful protocol and it may be coupled with many other applica- tions.

How is uracil converted to bisulfite in DNA?

The bisulfite modification technique uses bisulfite salt to deaminate cytosine residues on single-stranded DNA, converting them to uracil while leaving 5-methylcytosine unchanged (Figure 1). Figure 1. Example sequence of an unmethylated or methylated DNA template after bisulfite conversion. mC = methylated cytosine.