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Shedding light on DNA sequences
Pyrosequencing is a high-throughput sequencing-by-synthesis method used to quantify sequence variation. It’s based on a chemical reaction that generates a sequence of light signals, which reflects the sequence of the nascent DNA.
The pyrosequencing reaction relies on the action of four enzymes: DNA polymerase, ATP sulfurylase, luciferase and apyrase. DNA polymerase is used to elongate the DNA, upon which pyrophosphate is released. ATP sulfurylase converts the pyrophosphate to ATP, which is used by the luciferase to oxidize luciferin to oxyluciferin. This reaction generates a light signal, which is detected by a camera and displayed as peaks in a so called pyrogram. At the end of each reaction cycle, apyrase removes any unincorporated nucleotides left in the reaction.
The amount of light released in each pyrosequencing reaction, i.e. the height of each peak, is directly proportional to the number of incorporated nucleotides. If for example 3 nucleotides are incorporated, three ATPs are generated giving rise to triple peak in the pyrogram. The DNA sequence can be determined since the four nucleotides A, C, G and T are dispensed in a predefined order, so that each signal peak can be correlated with a specific nucleotide. This sequencing technology provides quantitative real-time data that can be used for characterization of single nucleotide polymorphisms (SNPs) and insertions-deletions (indels), as well as quantification of allele frequencies and DNA methylation levels.
For information about products based on this technology, see PyroMark.