How do you perform bisulfite sequencing?

How do you perform 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.

What does bisulfite sequencing detect?

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.

How do you quantify bisulfite converted DNA?

Quantification of Bisulfite-Converted DNA Converted DNA should be quantitated as RNA using a UV spectrophotometer (NanoDrop) with Ab260 nm 1.0 = 40 µg/ml.

What does bisulfite do to DNA?

Bisulfite Conversion is a process in which genomic DNA is denatured (made single-stranded) and treated with sodium bisulfite, leading to deamination of unmethylated cytosines into uracils, while methylated cytosines (both 5-methylcytosine and 5-hydroxymethylcytosine) remain unchanged.

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].

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.

How DNA methylation status is determined using bisulfite treatment?

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.

When was bisulfite sequencing invented?

These findings were later utilized by a group of Australian scientists to devise a means to analyze 5-methylcytosine in DNA; thus, a method called ‘bisulfite genomic sequencing’ was invented by these researchers in 1992.

What is the formula for bisulfite?

Sodium bisulfite/Formula

How much DNA is needed for bisulfite sequencing?

Number of clones submitted for sequencing depends on the application. Commonly 10–20 samples are sufficient, but for some applications as many as 50 may be necessary.

What is sodium bisulfite treatment used for?

Sodium bisulfite DNA treatment allows for discrimination between methylated and unmethylated cytosines.

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