For quantitative analysis of genetic or epigenetic DNA modifications using Pyrosequencing technology

  • Assay versatility on the same instrument and in the same run
  • Reliable quantification of allele representation and methylation status
  • Sequence information enables discovery of rare mutations
  • 1–24 samples can be analyzed in as little as 15 minutes
  • Compact detection platform uses minimal bench space

The PyroMark Q24 uses Pyrosequencing technology for real-time, sequence-based detection and quantification of sequence variants and epigenetic methylation. The PyroMark Q24 is highly suited for the analysis of CpG methylation, SNPs, insertion/deletions, STRs, variable gene copy number, as well as for microbial identification and resistance typing.

Quantity Product Cat. no. Price
 
 
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Quantity Product Cat. no. Price Sum
 
PyroMark Q24 System
Instrument and software for Pyrosequencing analysis: includes installation, training, and 1-year warranty on parts and labor
9001514
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PyroMark Q24 Priority Package
Instrument and software for Pyrosequencing analysis: includes Priority Package with installation, training, 2-year warranty on parts and labor, and 2 preventive maintenance visits
9001913
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PyroMark Q24 Priority Package Plus
Instrument and software for Pyrosequencing analysis: includes Priority Package Plus with installation, training, 3-year warranty on parts and labor, and 3 preventive maintenance visits
9001914
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PyroMark Q24 Software 2.0
Analysis software, for laboratory use only
9019062
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The PyroMark Q24 Instrument and PyroMark Q24 Software are intended to be used only in combination with QIAGEN kits indicated for use with the PyroMark Q24 for the applications described in the kit handbooks. If the PyroMark Q24 Instrument and PyroMark Q24 Software are used with other than QIAGEN kits, it is the user's responsibility to validate the performance of such product combination for any particular application.


CpG methylation analysis of the MLH1 gene.|Principle of Pyrosequencing — step 1.|Principle of Pyrosequencing — step 2.|Principle of Pyrosequencing — step 3.|Principle of Pyrosequencing — step 4.|Principle of Pyrosequencing — step 5.|CpG methylation pattern in the RASSF1A gene.|Workflow solutions.|Fully integrated system.|Easy data management.|Efficient template prep.|The right instrument for your needs.|
Highlighted areas in the Pyrogram trace indicate variable CpG positions (light gray) and built-in bisulfite treatment controls (yellow). The methylation level of each CpG site is indicated in blue boxes on top of the Pyrogram trace. Data kindly provided by Dr. Triantafillos Liloglou, Roy Castle Lung Cancer Foundation, Molecular Oncology, Liverpool.||||Apyrase, a nucleotide-degrading enzyme, continuously degrades unincorporated nucleotides and ATP. When degradation is complete, another nucleotide is added.||The figure illustrates the variation in methylation level between 5 different CpG sites in 4 individual tumor samples. Each sample was run in duplicate, and the concordance between the two samples clearly illustrates the reproducibility of Pyrosequencing technology.|The components of the PyroMark Q24 System are designed to make the Pyrosequencing workflow straightforward and efficient. Each step, from assay design to PCR amplification and preparation of sequencing templates, is supported by software, kits, reagents, and sample prep instrumentation optimized for Pyrosequencing.|Though small in size, the PyroMark Q24 manages all steps necessary to rapidly analyze up to 24 samples. Simply load your samples and reagents, upload your run file, and walk away. The PyroMark Q24 dispenses reagents and nucleotides to each well with precision. Light signals emitted are detected by 24 CCD sensors — one sensor per well — thereby eliminating signal crossover.|The PyroMark Q24 is designed as a stand-alone instrument, which makes it easy to place anywhere in the lab. Data are stored on the instrument hard drive and can be viewed on the instrument screen during a run. Additionally, all files are stored on the supplied USB stick, giving the user the flexibility to analyze data on any computer with PyroMark Q24 Software installed.|The PyroMark Q24 Vacuum Workstation enables conversion of PCR products into the single-stranded DNA for Pyrosequencing. Exposure of the PCR amplicons to a series of optimized solutions denatures and washes the DNA. This process is carried out for 24 samples in parallel and takes only a few minutes.|While the PyroMark Q24 combines ease-of-use, analysis versatility, and superior detection sensitivity, your workflow may demand higher throughput. The PyroMark Q96 ID and PyroMark Q96 MD accommodate this need by processing up to 96 samples in a single run. Furthermore, the automation options of the PyroMark Q96 MD enable hands-free processing of up to ten 96-well plates.|
Performance
A detection tool highly suited for epigenetics research

Pyrosequencing complements QIAGEN's epigenetics portfolio and enables accurate and sensitive quantification of methylation levels by providing highly reliable sequence data (see figure "CpG methylation analysis of the MLH1 gene"). It even allows the identification of novel mutations, as well as detection of aberrant DNA methylation patterns present at low levels. PyroMark Q24 includes a complete software package for CpG methylation analysis and a built-in control for bisulfite treatment.

Quantification of individual CpG sites

Analyzing individual CpG sites is crucial when studying differential gene expression in various tumors (see figure "CpG methylation pattern in the RASSF1A gene"). The lower resolution data provided by traditional methods fail to provide this degree of sensitivity. Pyrosequencing technology overcomes this challenge and enables analysis of single variations in the methylation pattern of single or multiple CpG sites with high accuracy.

Principle

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.

System component Description
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
PyroMark Q24 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.

Procedure
Fast and easy sample preparation 

From PCR product to single-stranded template ready for sequencing — up to 24 samples can be prepared in parallel using the PyroMark Q24 Vacuum Workstation, in less than 15 minutes. The workstation ensures easy handling, and the actual "hands-on time" is less than 5 minutes.

Prior to Pyrosequencing, a biotinylated PCR product is generated. This biotinylated PCR product is bound to Streptavidin-coated Sepharose beads, and the beads are captured with the Vacuum Tool on the Vacuum Workstation, where they are thoroughly washed and subsequently denatured, generating single-stranded DNA suitable for Pyrosequencing. This template DNA is released into the Pyrosequencing reaction plate containing the sequencing primer, and after primer annealing, the plate is placed into the PyroMark instrument. PyroMark Gold reagents contain the enzymes, nucleotides, and substrate for the Pyrosequencing reaction; these are pipetted into the dispensing tips or cartridge (depending on the instrument used), according to the volumes provided by the software, and are also placed into the instrument for the Pyrosequencing run.

Applications

Pyrosequencing is becoming increasingly important for research applications in a variety of disciplines. Whether examining drug-resistance development in pathogens, the role of epigenetic DNA methylation in gene expression regulation, genetic markers for specific phenotypes in livestock, or polymorphisms in forensic samples of mitochondrial DNA, the PyroMark Q24 enables powerful and versatile analysis of genetic and epigenetic variation. In addition, because Pyrosequencing integrates sequence detection and quantification, the enhanced analysis resolution can lead to new discoveries.

Software
Easy-to-use PyroMark Q24 software 

PyroMark Q24 software, installed on a PC, enables comprehensive analysis of your results. The software contains two analysis modes: CpG and AQ (allele quantification). Both modes can be used to analyze samples on the same plate, enabling different types of samples to be run at the same time. The AQ mode can be used for analyzing single and multivariable positions, as well as di-, tri- , and tetra- allelic mutations. The CpG mode enables analysis of multiple consecutive CpG sites and provides a built-in control for the bisulfite treatment.

Flexible and simple Pyrosequencing assay design using PyroMark Assay Design Software

PyroMark Assay Design Software 2.0 ensures easy design of PCR and sequencing primers. The assays are optimized for use with all PyroMark instruments.

Feature
Specifications
Altitude Up to 2000 m (6500 ft)
Applications Methylation analysis, allele quantification, genotyping, sequence analysis
Chemical resistance pH 4 to pH 9, common detergents, 0.5 M sodium hydroxide, ethanol
Connections One USB port (2.0)
Humidity Relative humidity of 20–90% (noncondensing)
Instrument dimensions 420 x 390 x 525 mm (16.5 x 15.4 x 20.7 in)
Kits designed for this instrument PyroMark Q24 Tests
Operating temperature 15–32°C (59–90°F)
Overvoltage category II
Place of operation For indoor use only
Pollution level 2
Power 100–240 V AC, 47–63 Hz, 1.1–0.45 A (grounded); from external power supply to the instrument : 12 VDC and 24 VDC nominal
Process temperature 28°C (82.4°F) ± 1%
Process time Depends on the number of dispensations (20 dispensations take 24 minutes)
Samples per run; throughput 1–24
Software PyroMark Q24 Software 2.0 (to be installed on PC)
Technology Pyrosequencing
Weight 27.5 kg (60.6 lb)

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