Pyrosekvensering

Pyrosekvensering, also known as pyrosequencing, is a method of DNA sequencing that relies on the detection of pyrophosphate released during DNA synthesis. It is a type of sequencing by synthesis technology. The process begins with the immobilization of single-stranded DNA fragments onto beads. These beads are then incubated with DNA polymerase, ATP, sulfurylase, and luciferase in the presence of dNTPs (deoxynucleotide triphosphates).

When a dNTP is incorporated into the growing DNA strand, pyrophosphate is released as a byproduct. Sulfurylase converts this pyrophosphate into ATP, which in turn fuels the luciferase enzyme to produce light. The intensity of the light emitted is proportional to the number of dNTPs incorporated at that position. By sequentially adding each of the four dNTPs (A, T, C, G) and measuring the light output at each step, the DNA sequence can be determined. Each cycle of dNTP addition and light detection reveals the presence and number of a specific nucleotide at a particular position in the template DNA.

Pyrosekvensering is known for its accuracy, particularly in detecting single nucleotide polymorphisms (SNPs) and short insertions or deletions. It was a popular method for targeted sequencing of specific genes or regions of interest before the widespread adoption of next-generation sequencing technologies. While its throughput is lower than modern platforms, its ability to generate high-quality reads and resolve homopolymer regions made it valuable for various research applications, including diagnostics and microbial identification. The technology has largely been superseded by more advanced sequencing platforms but remains a historically significant method in molecular biology.