[medhahegde]

Pyrosequencing or 454 sequencing (named after the Roche owned company that developed the method) is a novel, single nucleotide addition method (SNA method) of DNA sequencing and is increasingly utilized by the researchers nowadays. In this method, hybridization is achieved between a sequencing primer and a PCR amplicon (single stranded) that acts as a template. Following hybridization, the DNA is incubated with the enzymes such as DNA polymerase, apyrase, ATP sulfurylase and luciferase as well as with the substrates, luciferin and APS (adenosine 5' phosphosulfate).

The deoxribonucleotide triphosphates (dNTPs) are added sequentially. The enzyme DNA polymerase, catalyses the addition of deoxyribo-nucleotide triphosphate to the strand of DNA if it forms a complementary base to the base of the template strand. During each of the nucleotide incorporation, an equimolar quantity of pyrophosphate (ppi) to the incorporated nucleotide is released in the reaction.

The enzyme, ATP sulfurylase, catalyses the conversion of PPi to ATP in the presence of the substrate adenosine 5' phosphosulfate or APS. This ATP formed drives the conversion of luciferin to oxyluciferin mediated by luciferase enzyme that produces visible light in the proportional amount to that of ATP. This small amount of visible light produced by the action of enzymes as each nucleotide is incorporated to the growing chain is detected by CCD (charge coupled device) chip and is recorded as a series of peaks referred to as a pyrogram. The peaks correspond to the order of the nucleotides that are added revealing the underlying DNA sequence and the height of each peak is relative to the number of nucleotides incorporated.

Apyrase (nucleotide-degrading enzyme) degrades the unincorporated nucleotides and ATP continuously. And when the degradation is complete, a new nucleotide is added. Thus, by means of correlating the sample flashes with the nucleotide that is present at that time period, scientists are able to sequence a stretch of DNA.