PyroMark Q24 MDx

For IVD-validated mutation and methylation analysis using Pyrosequencing technology

Features

  • Compliant with EU IVD Directive 98/79/EC
  • Reliable quantification of allele representation and methylation status
  • Sequence information enables discovery of rare mutations
  • More than one assay can be completed in a single run
  • 1–24 samples can be analyzed in as little as 15 minutes

Products

The PyroMark Q24 MDx is intended for in vitro diagnostic use.

Product Details

The PyroMark Q24 MDx uses proven Pyrosequencing technology for real-time, sequence-based detection and quantification for in vitro diagnostic use in Europe. This innovative platform is highly suited for genotyping mutations, evaluating disease-related DNA methylation patterns, validating biomarkers, and other diagnostic-related assays.

Performance

Pyrosequencing technology enables accurate and sensitive quantification of genetic and epigenetic DNA variations by providing highly reliable sequence data. It allows the identification of novel mutations, as well as detection of aberrant DNA methylation patterns present at low levels.

The therascreen KRAS Pyro Kit is a good example of the informative analysis provided by the therascreen Pyro Kits and the PyroMark Q24 MDx instrument. The KRAS gene is mutated in approximately 35% of metastatic colorectal cancer (CRC) patients. Studies have shown that KRAS mutation testing can better define which CRC patients will benefit from treatment with epidermal growth factor receptor (EGFR) inhibiting monoclonal antibodies, such as panitumumab and cetuximab.

The therascreen KRAS Pyro Kit consists of 2 assays: one for detecting mutations in codons 12 and 13 and the other for detecting mutations in codon 61. The two regions are amplified separately by PCR, using optimized PCR reagents and primers included in the kit, and then sequenced through the defined region (see figure " Normal genotype in codons 12 and 13"). Sequences surrounding the defined positions serve as normalization and reference peaks for quantification and quality assessment of the analysis. Pyrosequencing technology on the PyroMark Q24 MDx enables identification of specific mutations (see figure " GGT to GAT mutation in base 2 of codon 12"), including less frequent mutations (see figures " GGT to AGT mutation in base 1 of codon 12" and " Reanalysis of the data in figure 'GGT to AGT mutation in base 1 of codon 12'"), as well as discovery of new mutations.

See figures

Principle

Pyrosequencing technology, which is based on the principle of sequencing by synthesis, provides quantitative data in sequence context within minutes.  PyroMark Q24 MDx is a fully integrated system that provides real-time sequence information and is highly suited for genetic and epigenetic analysis. The system includes the PyroMark Q24 MDx Instrument, PyroMark Q24 MDx Vacuum Workstation, PyroMark Q24 MDx Software 2.0, PyroMark Gold Q24 Reagents, PyroMark Control Oligo, and PyroMark Q24 Validation Oligo. Sample preparation solutions are also available to enable preparation of single-stranded DNA using the PyroMark Q24 MDx Vacuum Workstation.

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 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 elongated, and the nucleotide sequence is determined from the signal peaks in the Pyrogram trace (see figure " Principle of Pyrosequencing — step 5").

Dedicated IVD-validated assays

QIAGEN offers an expanding suite of IVD-validated assays to be used with the PyroMark Q24 MDx. Currently, these kits include important cancer-related mutations:

Product For quantitative measurement of mutations in
therascreen KRAS Pyro Kit codons 12, 13, and 61 of the human KRAS gene
therascreen BRAF Pyro Kit codons 600 and 464-469 of the human BRAF gene
therascreen EGFR Pyro Kit codons 719, 768, 790, 858, and 861 and exon 19 of the human EGFR gene
therascreen NRAS Pyro Kit codons 12, 13, and 61 of the human NRAS gene
See figures

Procedure

From PCR product to single-stranded template ready for sequencing — up to 24 samples can be prepared in parallel using the PyroMark Q24 MDx 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 cartridge, according to the volumes provided by the software, and are also placed into the instrument for the Pyrosequencing run.

Applications

The PyroMark Q24 MDx is suitable for in vitro diagnostic applications in Europe.

Genetic analysis comprises multiple applications to analyze differences in genomic DNA, including mutation detection and SNP typing. PyroMark Q24 MDx facilitates accurate and highly sensitive mutational analysis of any gene of interest and enables quantification of allele representation in mixed cell populations. The system can be used for self-validated assays for oncology studies and for analysis of epigenetic markers in methylation studies, or it can be used with dedicated IVD-validated assays available from QIAGEN

Software

PyroMark Q24 MDx 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.

Supporting data and figures

Specifications

FeaturesSpecifications
ConnectionsOne USB port (2.0)
Chemical resistancepH 4 to pH 9, common detergents, 0.5 M sodium hydroxide, ethanol
ApplicationsMethylation analysis, allele quantification, genotyping, sequence analysis
HumidityRelative humidity of 20–90% (noncondensing)
CE/FDA/IVD compatibleIn Europe
Instrument dimensions420 x 390 x 525 mm (16.5 x 15.4 x 20.7 in.)
Kits designed for this instrumentIVD-labeled therascreen Kits
Operating temperature15–32°C (59–90°F)
Overvoltage categoryII
Place of operationFor indoor use only
Pollution level2
Power100–240 V AC, 47–63 Hz, 1.1–0.45 A (grounded). From external power supply to instrument: 12 VDC and 24 VDC nominal
Process temperature28°C (82.4°F) ± 1°C
Process timeDepends on the number of nucleotide dispensations (20 dispensations take 24 minutes)
Samples per run (throughput)1–24
SoftwarePyroMark Q24 MDx Software 2.0
TechnologyPyrosequencing
Weight27.5 kg (60.6 lb)
AltitudeUp to 2000 m (6500 ft)

Resources

Download Files (15)
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0001
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0002
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0003
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0004
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0005
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0006
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0007
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0008
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0009
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0010
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0011
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0012
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0013
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0014
Instrument methods file for use with PyroMark Q24 MDx Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0015
Operating Software (3)
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 MDx software license and registered PyroMark Q24 MDx instrument. If you do not have a valid software license, contact your QIAGEN sales representative.

Publications

Pyrosequencing method to detect KRAS mutation in formalin-fixed and paraffin-embedded tumor tissues.
Dufort S; Richard MJ; de Fraipont F;
Anal Biochem; 2009; 391 (2):166-8 2009 May 21 PMID:19464247
Y chromosomal STR analysis using Pyrosequencing technology.
Edlund H; Allen M;
Forensic Sci Int Genet; 2009; 3 (2):119-24 2009 Jan 6 PMID:19215881
Amelogenin sex determination by pyrosequencing of short PCR products.
Tschentscher F; Frey UH; Bajanowski T;
Int J Legal Med; 2008; 122 (4):333-5 2008 Mar 20 PMID:18351373
Identification of mammal species using species-specific DNA pyrosequencing.
Karlsson AO; Holmlund G;
Forensic Sci Int; 2007; 173 (1):16-20 2007 Feb 28 PMID:17331687
More on contamination: the use of asymmetric molecular behavior to identify authentic ancient human DNA.
Malmström H; Svensson EM; Gilbert MT; Willerslev E; Götherström A; Holmlund G;
Mol Biol Evol; 2007; 24 (4):998-1004 2007 Jan 25 PMID:17255122

FAQ

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
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
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
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
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
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
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
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
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 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