What is bisulfite sequencing used for?

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.

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.

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 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.

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 is bisulfite method?

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.

What is the difference between CpG and FMCG?

CPG and FMCG products are regularly purchases, but FMCG is a subset of CPG , a product that just sell a bit faster. One of the most important differences between CPG and FMCG is the way we talk about sales.

What is the best bisulfite primer design tool?

Bisulfite Primer Seeker The Bisulfite Primer Seeker is a free bisulfite primer design tool that generates primers for Bisulfite Specific PCR (BSP). This free bisulfite primer design tool simplifies the tedious process of bisulfite primer design. Successful bisulfite primer design is critical to unbiased, region-specific DNA methylation analysis.

What are the considerations when starting with bisulfite conversion?

When starting with bisulfite conversion, there are a number of considerations to keep in mind to ensure optimal performance and subsequent accuracy of the DNA methylation analysis. Treatment with bisulfite is inherently a harsh process that dramatically changes both the chemical makeup and physical properties of the DNA.

How to improve the efficiency of bisulfite PCR?

The efficacy of bisulfite PCR is generally low; mispriming and non-specific amplification often occurs due to the T richness of the target sequences. In order to ameliorate the efficiency of PCR, we developed a new primer-design software called BiSearch, available on the World Wide Web.

What is bisulfite genomic sequencing?

Bisulfite genomic sequencing is the most widely used technique to analyze the 5-methylation of cytosines, the prevalent covalent DNA modification in mammals. The process is based on the selective transformation of unmethylated cytosines to uridines. Then, the investigated genomic regions are PCR amplified, subcloned and sequenced.