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 DOCTORAL RESEARCH

Dissertation Title: Novel DNA sequencing technology “Pyrosequencing: Enzymes & Assays”

I completed my Ph.D. in the Department of Biochemistry and Biotechnology at the Royal Institute of Technology (Stockholm, Sweden) in the laboratory of Dr. Pål Nyrén . As part of my graduate work, I participated in the development of a novel real-time DNA sequencing technology, also known as ”Pyrosequencing”.

 

Pyrosequencing is a novel real-time DNA sequencing technology evolved to the formation of a company “Biotage” which markets equipment for DNA sequencing and SNP genotyping.

The Principle of Pyrosequencing

 

Step 1
A sequencing primer is hybridized to a single stranded, PCR amplified, DNA template, and incubated with the enzymes, DNA polymerase, ATP sulfurylase, luciferase and apyrase, and the substrates, adenosine 5´ phosphosulfate (APS) and luciferin.

 

Step 2
The first of four deoxynucleotide triphosphates (dNTP) is added to the reaction. DNA polymerase catalyzes the incorporation of the deoxynucleotide triphosphate into the DNA strand, if it is complementary to the base in the template strand. Each incorporation event is accompanied by release of pyrophosphate (PPi) in a quantity equimolar to the amount of incorporated nucleotide.

 

Step 3
ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5´ phosphosulfate. This ATP drives the luciferase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by a charge coupled device (CCD) camera and seen as a peak in a pyrogram™. Each light signal is proportional to the number of nucleotides incorporated.


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Step 4
Apyrase, a nucleotide degrading enzyme, continuously degrades unincorporated dNTPs and excess ATP. When degradation is complete, another dNTP is added.

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Step 5
Addition of dNTPs is performed one at a time. It should be noted that deoxyadenosine alfa-thio triphosphate (dATPalphaS) is used as a substitute for the natural deoxyadenosine triphosphate (dATP) since it is efficiently used by the DNA polymerase, but not recognized by the luciferase.

As the process continues, the complementary DNA strand is built up and the nucleotide sequence is determined from the signal peak in the pyrogram.


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