454 pyrosequencing is a method of high throughput DNA sequencing that utilizes a single strand of DNA with a length of 400-500bp. The single strand is used as a template to synthesize the sequence of its complementary strand, which is determined by a chain of reactions resulting in light being emitted when a specific nucleotide or length of nucleotides are added to the complementary sequence. This method was originally developed at the Royal Institute of Technology in Stockholm, Sweden in 1996. 454 life sciences adapted pyrosequencing for high throughput sequencing knows as 454 pyrosequencing by incorporating an array based pyrosequencing technology. The array based method allows sequencing of 400-600 megabases in a 10 hour period allowing an entire human genome to be sequenced in ~27 days.
Sequencing Method[]
Pyrosequencing requires 4 enzymes, dNTP's, 2 substrates (adenosine 5' phosphosulfate (APS) & luciferin), a single strand of DNA and a light sensitive camera. The 4 required enzymes are:
DNA Polymerase: used to elongate complementary DNA strand.
ATP Sulfurylase: converts pyrophosphate (PPi) into ATP in the presence of adeosine 5' phosphosulfate (APS).
Luciferase: Uses ATP to convert luciferin to oxyluciferin, which is a molecule that emits visible light.
Apyrase: Degrades unincorporated nucleotides.
The sequencing process is as follows:
A 400-500bp ssDNA is incubated with DNA Polymerase, ATP Sulfurylase, Luciferase, Apyrase, Luciferin and APS.
Pyrosequencing
Pyrosequencing Animation from QIAGEN
A splash of one of the four dNTP's is added to the incubation mentioned above, the polymerase will elongate the complementary strand if the dNTP added pairs with the ssDNA template. If the dNTP does not pair with the ssDNA, it is digested by the apyrase.
If the dNTP pairs with the ssDNA the polymerase elongates the complementary strand which causes the release of PPi (pyrophosphate).
The ATP Sulfurylase then converts the 1 PPi and 1 APS substrate to ATP. (1 PPi+ 1 APS= 1 ATP)
ATP catalyses the Luciferase reaction that converts the substrate luciferin to oxyluciferin.
The oxyluciferin emits light that can be captured with a very sensitive c
Pyrosequencing sequence output chart. QIAGEN
amera and analyzed in a program to produce a graph showing when light was captured and how much light was captured in relation to which dNTP was added to the incubation. If a dNTP does not match, no light is emitted.
Any remaining dNTP's are digested by the apyrase, to provide a pure environment for the next dNTP.
As mentioned above the oxyluciferin will emit different levels of light. If the light is brighter, the computer program analyses how many dNTPs were paired in tandem to the ssDNA.
454 Life Sciences[]
Sequencing: One Bead = One Read Load the beads onto the PicoTiterPlate device, where the surface design allows for only one bead per well. The PTP Device is then loaded in instrument for sequencing. Individual nucleotides are flowed in sequence across the wells. Each incorporation of a nucleotide complementary to the template strand results in a chemiluminescent light signal recorded by the camera.
454 life sciences improved upon this method by creating pyrosequencing assays. Short, identical segments of ssDNA are wrapped around a DNA capturing bead and then placed in a well of the microassay where it is subject to PCR with the enzymes and substrates mentioned above. This allows many ssDNA segments to be sequenced at once.
