Pyrosequencing technology, which is based on the principle of sequencing by synthesis, provides quantitative data in sequence context within minutes. PyroMark Q24 is a fully integrated system that provides real-time sequence information, and is highly suitable for epigenetic research and genetic analysis. The system includes PyroMark Q24, PyroMark Q24 Vacuum Workstation, PyroMark Q24 Software, PyroMark Gold Q24 Reagents, PyroMark Control Oligo, and PyroMark Q24 Validation Oligo (see table). Sample preparation solutions are also supplied to enable preparation of single-stranded DNA using the PyroMark Q24 Vacuum Workstation.
|PyroMark Q24 ||Sequencing instrument for quantitative mutational and methylation analysis |
|PyroMark Q24 Vacuum Workstation ||Workstation for sample preparation of up to 24 samples in parallel |
|PyroMark Q24 Software ||Analysis software; provided in 2 analysis modes (for CpG analysis and allele quantification) |
|PyroMark Q24 Gold Q24 Reagents ||Enzymes, substrates, and nucleotides |
|PyroMark Q24 Control Oligo ||Control for verification of proper installation and operation of the system |
|PyroMark Q24 Validation Oligo ||Control for performance confirmation of the system |
A highly suitable platform for genetic analysis
Genetic analysis comprises multiple applications to analyze differences in genomic DNA, including mutation detection and SNP typing. PyroMark Q24 facilitates accurate and highly sensitive mutational analysis of any gene of interest, and enables quantification of allele representation in mixed cell populations. QIAGEN also offers optimized and validated RUO tests for analyzing particular gene mutations by Pyrosequencing.
Steps of the Pyrosequencing reaction:
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. 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 — step 1").
Step 2: The first deoxribonucleotide triphosphate (dNTP) is added to the reaction. DNA polymerase catalyzes the addition of the dNTP to the squencing primer, 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 (see figure " Principle of Pyrosequencing — step 2").
Step 3: 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 CCD sensors 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 3").
Step 4: 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 4").
Step 5: Addition of dNTPs is performed sequentially. It should be noted that deoxyadenosine alpha-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 elongated, and the nucleotide sequence is determined from the signal peaks in the Pyrogram trace (see figure " Principle of Pyrosequencing — step 5").
Streamlined workflow — from sample to result
The versatile PyroMark Q24 seamlessly integrates into epigenetics and genetic analysis workflows, and complements QIAGEN's advanced technologies for sample preparation, bisulfite conversion, and PCR amplification. The highly reliable instrument enables sequence-based detection and quantification of CpG sites as well mutations. The streamlined workflow means that results can be achieved faster.