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PyroMark Q24

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

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PyroMark Q24 System

Cat. No. / ID: 9001514

Instrument and software for Pyrosequencing analysis: includes installation, training, and 1-year warranty on parts and labor
PyroMark Q24
PyroMark Q24 Software
This product will be discontinued as of December 31, 2025 or until stocks last.
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.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Product Details

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.

Explore the virtual demo to learn more about the PyroMark Q24.


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.

See figures


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.

See figures


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.


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.


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.

Supporting data and figures

Service Plans

Pyro Q24, Full Agreement

Cat. No. / ID: 9241822

Instrument repair service for the PyroMark Q24 at regional repair center. Shipping, labor and parts included during the Full Agreement period. Loaner instrument provided within 2–3 business days. Instrument repair turnaround time of 7–10 business days. Includes one on-site Preventive Maintenance or Inspection Service during the Full Agreement period.
Pyro Q24, Preventive Subscription

Cat. No. / ID: 9243547A

One on-site Preventive Maintenance or Inspection Service visit for the PyroMark Q24, including travel, labor and parts. Includes a 10% discount on repair services during the Preventive Subscription period.


Operating Software (1)
This version is compatible with Windows 7 and Windows 10 (64 bit) operating systems. This software may only be downloaded by registered users with a valid PyroMark Q24 software license and registered PyroMark Q24 instrument. If you do not have a valid software license, contact your QIAGEN sales representative.
Application Notes (3)
Download Files (15)
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0008
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0013
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0004
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0002
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0009
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0011
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0010
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0005
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0003
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0012
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0001
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0006
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0007
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0014
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0015
Instrument User Manuals (1)
Software User Guides (1)


Genetic diagnostics of functional variants of the human dopamine D2 receptor gene.
Doehring A; Kirchhof A; Lötsch J;
Psychiatr Genet; 2009; 19 (5):259-68 2009 Oct PMID:19512960
Assessing combined methylation-sensitive high resolution melting and pyrosequencing for the analysis of heterogeneous DNA methylation.
Candiloro IL; Mikeska T; Dobrovic A;
Epigenetics; 2011; 6 (4):500-7 2011 Apr 1 PMID:21364322
Cell specific patterns of methylation in the human placenta.
Grigoriu A; Ferreira JC; Choufani S; Baczyk D; Kingdom J; Weksberg R;
Epigenetics; 2011; 6 (3):368-79 2011 Mar 1 PMID:21131778
CpG island hypermethylation of the neurofibromatosis type 2 (NF2) gene is rare in sporadic vestibular schwannomas.
Kullar PJ; Pearson DM; Malley DS; Collins VP; Ichimura K;
Neuropathol Appl Neurobiol; 2010; 36 (6):505-14 2010 Oct PMID:20831745
Systematic cross-validation of 454 sequencing and pyrosequencing for the exact quantification of DNA methylation patterns with single CpG resolution.
Potapova A; Albat C; Hasemeier B; Haeussler K; Lamprecht S; Suerbaum S; Kreipe H; Lehmann U;
BMC Biotechnol; 2011; 11 :6 2011 Jan 14 PMID:21235780


Where can I order the Streptavidin Sepharose beads for pyrosequencing?
The recommended Streptavidin Sepharose High Performance beads for pyrosequencing can be ordered at GE healthcare with the catalog no 17-5113-01.

The PyroMark Q48 Autoprep protocol uses magnetic streptavidin-coated Sepharose® beads (PyroMark Q48 Magnetic Beads), which bind to the biotinylated PCR strand.

PyroMark Q48 Magnetic Beads can be ordered at QIAGEN with the catalog no 974203.

FAQ ID -2850
Can I order the nucleotides from PyroMark Gold Reagents separately?
The nucleotides can only be ordered as part of the PyroMark Gold Reagents which also contain enzyme and substrate mix.
FAQ ID -2827
How many nucleotides of a homopolymer can be resolved in pyrosequencing?
In the range of 3-5 bases can be resolved depending on the sequence context and base. If it is possible sequencing of a homopolymer of more than 3-5 nucleotides should be avoided by resetting the sequencing primer.
FAQ ID -2871
How much space does the PyroMark Q24 instrument need?

PyroMark Q24 takes very little bench space, measuring only H420 x W390  x D525 mm.


FAQ ID -2098
What is a PyroMark instrument method or instrument code?

An instrument method or instrument code encodes the individual pulse time settings of specific cartridge lot batch. These pulse time settings change when e.g. a new batch of capillaries is used with slight variations in the needle diameter. For larger diameters, the pulse settings are lowered to dispense the correct volume of liquid. In addition, the viscosity of enzyme and substrate mixes can change which influences dispensing volumes.

The individual instrument method/code number is printed on the cartridge label. The corresponding methods/code settings can be downloaded as a file from the respective instrument webpage and opened in the PyroMark application software.

FAQ ID -2941
What is the reason for a high substrate peak in the pyrosequencing pyrogram?
Usually pyrophosphate or dATP/ATP contamination in the sample or in the buffer can cause a high substrate peak. Large amounts of pyrophosphate are generated in the PCR reaction and might be carried over to the sequencing reaction. Check the PyroMark buffers and reagents and use new ones.
FAQ ID -2879
How does the built-in QC control for complete bisulfite conversion of DNA work on PyroMark Q24?

Data generated with the methylation analysis software (detection and quantification of CpG sites) on PyroMark Q24 contains unique features that act as quality control for complete bisulfite conversion of DNA. When the assay encounters a C not followed by a G (C unmethylated), that C should be fully converted to T if the bisulfite treatment upfront was successful. Subsequently, it should be presented in the pyrogram as T=100%. This acts as a useful quality control for full conversion of unmethylated C residues during bisulfite treatment and PCR.

FAQ ID -2095
Which end of the PCR primer for pyrosequencing should be biotinylated?
In Pyrosequencing, the 5' end should be biotinylated, regardless of whether the forward or reverse primer is biotinylated. You can order pyrosequencing primers here.
FAQ ID -2839
What kind of shaker should be used for the pyrosequencing binding step?
Shaking conditions are 1400 rpm at room temperature. Optimal results are obtained with 2mm orbital diameter.
FAQ ID -2837
What should be the single peak height for the PyroMark Control Oligo on the different PyroMark instruments?

PyroMark Q24: The mean single peak height is 95 +/- 55 RLU
PyroMark Q48: The mean single peak height is 70 +/- 40 RLU
PyroMark Q96 ID: The mean single peak height is 35 +/- 10 RLU
Pyromark Q96 MD: The mean single peak height should be at least 350 RLU

FAQ ID -2852
What is the use of the PyroMark Q24 Validation Oligo?
The PyroMark Q24 Validation Oligo was developed to check the performance of the PyroMark Q24 system. It consists of two biotinylated oligonucleotides that only differ in one position (A or G) of the sequence. By mixing different proportions of the two oligonucleotides in replicates the linearity, bias and reproducibility of the system can be determined.
FAQ ID -2855
What is the sensitivity limitation for pyrosequencing?
In general, the standard claim for pyrosequencing sensitivity is about 5% which is also published in many papers. The actual sensitivity limit is assay dependent and has to be determined individually.
FAQ ID -2840
When do I have to change the pulse settings/methods in a pyrosequencing run setup?
Always check for the actual method/code number printed on the cartridge label. Make sure that you choose this method/code number when setting up the pyrorun in the application software. If this method cannot be selected automatically in the application software, you can download the method/code file from the instrument webpage.
FAQ ID -2942
We recently changed the OS from Windows XP to Windows 7. When re-installing software Pyromark Q24 2.0.6, it fails to analyse the results. Any suggestions?

Please install software 2.0.7, which is compatible with Windows 7, 64 and 32 bit.

FAQ ID - 3621
How do I reduce background peaks in the pyrosequencing pyrogram?
There are several reasons for a high assay background; the template can form secondary structures which are extended or the primers itself form dimmers which serve as template. Perform accurate sequencing controls (e.g. PCR or sequencing primer only) as recommended in the PyroMark User Manual to observe this kind of background. In addition, an unspecific priming of primer to template or unspecific annealing of sequencing primer to template might also be a background cause. Please check your complete primer design and if needed, perform a redesign. Try to lower the primer concentration as possible to avoid excess of primer.
FAQ ID -2877
What is the sample throughput of Pyrosequencing systems?

PyroMark instruments offer a range of throughput scales. The PyroMark Q24 can process 1–24 samples in parallel, the PyroMark Q48 Autoprep 1-48, the PyroMark Q96 ID 1–96, and the PyroMark Q96 MD 1-96, or the automation option enables automated processing of ten 96-well plates. The sample processing speed depends on the number of nucleotide dispensations necessary for the programmed analysis. Twenty dispensations take approximately 24 minutes on all instruments; thus, 96 samples are typically processed in 10-100 minutes.



FAQ ID -2215
What is the concentration of PyroMark Control Oligo?
PyroMark Control Oligo has a concentration of 20µM and is delivered in a volume of 50µl. Two tubes of 10x dilution buffer (2x 1.7ml) are delivered with the control oligo.
FAQ ID -2846
How many times can vacuum troughs be re-used with the PyroMark Vacuum Preparation Stations?
There is no precise recommendation how many times these troughs on the PyroMark Vacuum Preparation Stations (Q24 and Q96) can be re-used. It depends on the individual handling and cleaning (with water).
FAQ ID -2848
Which heating block is recommended for the pyrosequencing annealing step?
A heating block that can heat up to 80-90 °C is recommended. A solid block is preferable. For the PyroMark Q24 the surface area must be 50mm x 60 mm and for Pyromark Q96 ID/MD 120mm x 80 mm.
FAQ ID -2838
Can I reinstall the PyroMark Q24 software on my new computer or following an operating system upgrade, or do I need to purchase a new license?

If you need to install the PyroMark Q24 software on a new computer replacing your old one, or after you reinstall or upgrade the operating system, you can reinstall the PyroMark Q24 software without purchasing a new license.

FAQ ID - 3473

What concentration should be used for the sequencing primer in pyrosequencing?

Usually the sequencing primer is used at 0.3µM in annealing buffer but some assays might require additional optimization of the sequencing primer concentration.


For PyroMark Q24 and PyroMark Q96 MD the final concentration of the sequencing primer is 0.3µM and for PyroMark Q96 ID 0.4µM.

The PyroMark Q48 Autoprep dispenses the sequencing primers for annealing. The final concentration of sequencing primers in a well is 0.8µM, but may be adapted to optimize assays.


FAQ ID -2826
Where to find explanations to the warning given by the PyroMark software after run data analysis?
The PyroMark Q24 application software contains under Help (Press F1) a Pyromark Q24 Software user guide with all essential information about warnings and software features. The Pyromark ID/MD software also contains a softwareware guide under help. Moreover, the individual instrument user manuals contain helpful information in the troubleshooting section.
FAQ ID -2874
Which purity grade is recommended for pyrosequencing primers?
Only the biotinylated primer needs to be HPLC purified whereas the other primers require standard desalting only.  Pyrosequencing primers can be ordered here.
FAQ ID -2832
What is the reason for split peaks appearing in between dispensations on my pyrosequencing pyrogram?
The PyroMark cartridge needle can be blocked or damaged. Clean the cartridge or exchange with a new one. Check for correct reagent cartridge and cartridge method used in the run. Check if the reagent cartridge cover was closed properly. Make sure that the cartridge was dry after cleaning because nucleotide droplets might be caught at the needle tip and fall down at any time. or exchanged.
FAQ ID -2881
Will dUTP in a PCR reaction affect pyrosequencing?
In general, dUTP/UNG treatment should work for pyrosequencing in order to reduce contamination risk with PCR amplicons from previous PCR’s.
FAQ ID -2843
Is the CpG software included in the PyroMark instruments to study methylation status?
The PyroMark Q24 software and the new PyroMark Q96 ID software version 2.5 support CpG analysis in the CpG mode. The ORACLE-based PyroMark Q96 ID software version and PyroMark Q96 MD software do not support CpG analysis. In this case an independent, additional software is needed which is the PyroMark CpG software version 1.0.
FAQ ID -2842
How long does a single run on the PyroMark Q24 take?

PyroMark Q24 analyses up to 24 individual samples in a single run. The time needed to perform a run depends on the number of nucleotides dispensed in the specific assay. A typical SNP assay contains 15 dispensations and a typical CpG assay 45. Since the estimated dispensation time is 1 minute/nucleotide, a typical SNP run takes 15 minutes to perform and a typical CpG assay takes 45 minutes.

FAQ ID -2096
What is the reason for signals ceasing in the middle of a pyrosequencing run?
The cartridge needle can be blocked or damaged causing a dispensation error. Clean the cartridge following the guidelines or repeat the run with a new cartridge. On the other hand if high amounts of template have been used resulting in very high signals (>100 RLU), the substrate for the sequencing reaction might be depleted. In this case template conditions should be optimized.
FAQ ID -2875
What kind of reading length can I expect when using Pyrosequencing technology for sequence analysis?

Typical reading length using Pyrosequencing technology is 40-60 bases. However, as with any sequencing technology, the maximum read length will depend on template secondary structure, base content, quality of PCR-product, and other parameters.

Depending on the sequence to be analyzed, highly accurate read lengths of 140 or more bases can be obtained in just a single reaction with the Q48 PyroMark Autoprep.



FAQ ID -2216
What is the recommended amplicon size for CpG assays?
The amplicon length should be short (<200bp). This is critical especially for DNA from FFPE tissue which is often degraded by the fixation so that short fragments are easier to amplify. Moreover, the DNA suffers from harsh bisulfite treatment and might receive further double strand breaks. Therefore the amplicon size should be kept as short as possible.
FAQ ID -2825
Can unused wells in a pyrosequencing plate be used in the next run?
In principle it’s possible to use so far unused pyrosequencing wells for the next run and leave the already used wells empty. However, due to contamination risk when cleaning and handling plates QIAGEN does not recommend this.
FAQ ID -2872
How accurate and reliable is PyroMark Q24 in mutation analysis?

PyroMark Q24 uses Pyrosequencing technology for mutation analysis and provides a built-in quality control in each run. By sequencing nucleotides flanking the mutation of interest, the researcher gets confirmation that the assay was made at the correct position. In addition, blank dispensations are included in the assay providing a negative control of the run. 

FAQ ID -2094
How do I prevent a drifting baseline in my pyrosequencing pyrogram?
Let the PyroMark instrument warm up (about 60 minutes) to adapt to room temperature before use. Make sure the ambient room temperature is within range 18-28°C.
FAQ ID -2878