PYROSEQUENCING

Pyrosequencing is

method

of DNA

sequencing (determining

the

order

of nucleotides in DNA) based on the "sequencing by synthesis" principle. It differs fromSanger sequencing, in that it relies on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with dideoxynucleotides.[1]

The technique was developed by Pl Nyrn and Mostafa Ronaghi at the Royal Institute of Technology in Stockholm in 1996.[2][3][4] The desired DNA sequence is able to be determined by light emitted upon incorporation of the next complementary nucleotide by the fact that only one out of four of the possible A/T/C/G nucleotides are added and available at a time so that only one letter can be incorporated on the single stranded template (which is the sequence to be determined). The intensity of the light determines if there are more than one of these "letters" in a row. The previous nucleotide letter (one out of four possible dNTP)is degraded before the next nucleotide letter is added for synthesis: allowing for the possible revealing of the next nucleotide(s) via the resulting intensity of light (if the nucleotide added was the next complementary letter in the sequence). This process is repeated with each of the four letters until the DNA sequence of the single stranded template is determined. Procedure "Sequencing by synthesis" involves taking a single strand of the DNA to be sequenced and then synthesizing its complementary strand enzymatically. The pyrosequencing method is based on detecting the activity of DNA polymerase (a DNA synthesizing enzyme) with another chemiluminescent enzyme. Essentially, the method allows sequencing of a single strand of DNA by synthesizing the complementary strand along it, one base pair at a time, and detecting which base was actually added at each step. The template DNA is immobile, and solutions of A, C, G, and T nucleotides are sequentially added and removed from the reaction. Light is produced only when the nucleotide solution complements the first unpaired base

of the template. The sequence of solutions which produce chemiluminescent signals allows the determination of the sequence of the template. ssDNA template is hybridized to a sequencing primer and incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase and apyrase, and with the substrates adenosine 5 phosphosulfate (APS) and luciferin. 1. The addition of one of the four deoxynucleoside triphosphates (dNTPs) (dATPS, which is not a substrate for a luciferase, is added instead of dATP) initiates the second step. DNA polymerase incorporates the correct, complementary dNTPs onto the template. This incorporation

releases pyrophosphate (PPi) stoichiometrically. 2. ATP sulfurylase quantitatively converts PPi to ATP in the presence of adenosine 5 phosphosulfate. This ATP acts as fuel to 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 camera and analyzed in a program. 3. Unincorporated nucleotides and ATP are degraded by the apyrase, and the reaction can restart with another nucleotide.

Currently, a limitation of the method is that the lengths of individual reads of DNA sequence are in the neighborhood of 300-500 nucleotides, shorter than the 800-1000 obtainable with chain terminationmethods (e.g. Sanger sequencing). This can make the process of genome assembly more difficult, particularly for sequences containing a large amount of repetitive DNA. As of 2007, pyrosequencing is most commonly used for resequencing or sequencing of genomes for which the sequence of a close relative is already available.

The templates for pyrosequencing can be made both by solid phase template preparation (streptavidin-coated magnetic beads) and enzymatic template

preparation (apyrase+exonuclease).

: Pyrosequencing is a DNA sequencing technique that is based on the detection of released pyrophosphate (PPi) during DNA synthesis. In a cascade of enzymatic reactions, visible light is generated that is proportional to the number of incorporated nucleotides (Fig.1). The cascade starts with a nucleic acid polymerization reaction in which inorganic PPi is released as a result of nucleotide incorporation by polymerase. The released PPi is subsequently converted to ATP by ATP sulfurylase, which provides the energy to luciferase to oxidize luciferin and generate light. Because the added nucleotide is known, the sequence of the template can be determined. The nucleic acid molecule can be either RNA or DNA. However, because DNA polymerases show higher catalytic activity than RNA polymerases for limited nucleotide extension, efforts have been focused on the use of a primed DNA template for pyrosequencing. Standard pyrosequencing uses the Klenow fragment of Escherichia coli DNA Pol I, which is a relatively slow polymerase (Benkovic and Cameron 1995). The ATP sulfurylase used in pyrosequencing is a recombinant version from the yeast Saccharomyces cerevisiae(Karamohamed et al. 1999a) and the luciferase is from the American firefly Photinus pyralis. The overall reaction from polymerization to light detection takes place within 34 sec at room temperature. One pmol of DNA in a pyrosequencing reaction yields 61011 ATP molecules which, in turn, generate more than 6109 photons at a wavelength of 560 nanometers. This amount of light is easily detected by a photodiode, photomultiplier

tube, or a charge-coupled device camera (CCD) camera. There are two different pyrosequencing strategies that are currently available: solid-phase pyrosequencing (Ronaghi et al. 1996) and liquid-phase pyrosequencing (Ronaghi et al. 1998b). Solidphase pyrosequencing (Fig. 2) utilizes immobilized DNA in the three-enzyme system described previously. In this system a washing step is performed to remove the excess substrate after each nucleotide addition. In liquid-phase pyrosequencing (Fig.3) apyrase, a nucleotide-degrading enzyme from potato, is introduced to make a four-enzyme system. Addition of this enzyme has eliminated the need for solid support and intermediate washing thereby enabling the pyrosequencing reaction to be performed in a single tube.

These formats are described in detail in this review

Step

A sequencing primer is hybridized to a singlestranded PCR amplicon that serves as a template, and incubated with the enzymes, DNA polymerase, ATP sulfurylase, luciferase, and apyrase as well as the substrates, adenosine luciferin. 5' phosphosulfate (APS), and

Step The first deoxribonucleotide

2 triphosphate

(dNTP) is added to the reaction. DNA polymerase catalyzes the incorporation of the deoxyribo-nucleotide 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

ATP sulfurylase converts PPi to ATP in the presence of adenosine 5' phosphosulfate (APS). 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) chip and seen as a peak in the raw data output

(Pyrogram). The height of each peak (light signal) is proportional to the number of nucleotides incorporated.

Step Apyrase, a nucleotide-degrading degrades

4 enzyme,

continuously

unincorporated

nucleotides and ATP. When degradation is complete, another nucleotide is added.

Step

Addition of dNTPs is performed sequentially. It should be noted that deoxyadenosine alfathio triphosphate (dATPS) 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 peaks in the Pyrogram trace.