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.
What is bisulfite treatment?
Bisulfite Conversion is a method used to make it easy to distinguish methylated from unmethylated cytosines in genomic DNA at single base resolution. DNA is first denatured (made single-stranded) and then treated with sodium bisulfite.
How is 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.
What is DNA pyrosequencing?
Pyrosequencing is a method of DNA sequencing that differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release and the generation of light on nucleotide incorporation, rather than chain termination with dideoxynucleotides.
Is pyrosequencing next generation sequencing?
Pyrosequencing, developed by 454 Life Sciences, was one of the early successes of Next-generation sequencing; indeed, 454 Life Sciences produced the first commercially available Next-generation sequencer.
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.
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 is DNA sequenced by pyrosequencing?
Pyrosequencing is a method of DNA sequencing (determining the order of nucleotides in DNA) based on the “sequencing by synthesis” principle, in which the sequencing is performed by detecting the nucleotide incorporated by a DNA polymerase.
What is the difference between pyrosequencing and Sanger sequencing?
The main difference between Sanger sequencing and pyrosequencing is that Sanger sequencing is a DNA sequencing approach that uses the dideoxy chain termination method, whereas pyrosequencing is a DNA sequencing approach based on the sequencing-by-synthesis principle.