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 |