Pyrosequencing Technology and Platform Overview

Pyrosequencing reaction cascade
Pyrosequencing — the unique detection and quantification technology
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Characterization of complex DNA modifications underlying gene expression patterns requires quantifiable sequence data. Pyrosequencing is a sequence-based detection technology that enables rapid and accurate quantification of sequence variation. Streamlined protocols, analysis flexibility, and elegant output make Pyrosequencing technology a highly adaptable tool for exploratory and testing work in a broad range of disciplines.
Principle of Pyrosequencing
Comparison of PyroMark platforms
Principle of Pyrosequencing
Step 1
A DNA segment is amplified and the strand to serve as the Pyrosequencing template is biotinylated. After denaturation, the biotinylated single-stranded PCR amplicon is isolated and allowed to hybridize with a sequencing primer (see figure Principle of Pyrosequencing — steps 1–3).

Step 2 
The hybridized primer and single-stranded template are incubated with the enzymes DNA polymerase, ATP sulfurylase, luciferase, and apyrase, as well as the substrates adenosine 5' phosphosulfate (APS) and luciferin (see figure Principle of Pyrosequencing — steps 1–3).

Step 3 
The first deoxyribonucleotide triphosphate (dNTP) is added to the reaction. DNA polymerase catalyzes addition of the dNTP to the sequencing primer, if it is complementary to the base in the template strand. Each incorporation event is accompanied by the release of pyrophosphate (PPi) in a quantity equimolar to the amount of incorporated nucleotide (see figure Principle of Pyrosequencing — steps 1–3).

Step 4
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) camera 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 (see figure Principle of Pyrosequencing — step 4).

Step 5
Apyrase, a nucleotide-degrading enzyme, continuously degrades unincorporated nucleotides and ATP. When degradation is complete, another nucleotide is added (see figure Principle of Pyrosequencing — step 5).

Step 6
Addition of dNTPs is performed sequentially. It should be noted that deoxyadenosine alfa-thio triphosphate (dATPαS) 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 (see figure Principle of Pyrosequencing — step 6).
Comparison of PyroMark platforms
  PyroMark Q48 Autoprep PyroMark Q24 Advanced PyroMark Q24  PyroMark Q96 ID
Main applications Complex mutation analysis
Epigenetics (CpG and CpN analysis)
Resistance typing and microbial ID  
Complex mutation analysis
Epigenetics (CpG and CpN analysis)
Resistance typing and microbial ID  
Mutation analysis
Resistance typing 
 
Mutation analysis
Epigenetics
Resistance typing and microbial ID 
Throughput 1–48 samples 1–24 samples 1–24 samples 1–96 samples
Running volume 10 µl 25 µl 25 µl  40 µl 
PCR requirements 5–10 µl
(~0.5–3 pmol of product)
5–10 µl
(~0.5–3 pmol of product)
5–10 µl
(~0.5–3 pmol of product)            
20–40 μl
(2–4 pmol of product)
Maximum read length 10–140* bp or more 10–140* bp or more 10–80* bp 10–80* bp 
Template preparation automated manual manual manual
Application software PyroMark Q48 Autoprep SW
PyroMark Q24 Advanced SW
(requires firmware 1.5.6903 or higher)
PyroMark Q24 SW 2.0 PyroMark Q96 SW
Software functionality SEQ (de novo sequencing)
CpG/CpN methylation
SNP
AQ
SEQ (de novo sequencing)
CpG/CpN methylation
SNP
AQ
SQA (de novo sequencing)
CpG methylation
AQ/SNP 
 
SQA (de novo sequencing) 
CpG methylation
SNP
AQ
Compatible reagents PyroMark Q48 Advanced Reagents
PyroMark Q48 Advanced CpG Reagents  
PyroMark Q24 Advanced Reagents
PyroMark Q24 Advanced CpG Reagents  
PyroMark Q24 Gold Reagents  PyroMark Gold Q96 Reagents 
Sensitivity 2% mutation
98% wt  
2% mutation
98% wt  
2% mutation
98% wt  
2% mutation
98% wt  
* Actual read length varies, depending on the specific assay and sequence being analyzed.
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