PyroMark Q24 MDx Accessories and Reagents

For use with the PyroMark Q24 MDx

Features

  • CE-marked as compliant with EU IVD Directive 98/79/EC
  • Highly accurate results for in vitro diagnostics
  • Reliable quantification of allele representation
  • Sequence information enables discovery of rare mutations
  • 1–24 samples can be analyzed in as little as 15 minutes

Products

PyroMark Q24 MDx Accessories are intended for in vitro diagnostic use.
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PyroMark Vacuum Prep Filter Probe (100)

Cat. No. / ID: 979010

Reusable filter probes for PyroMark Vacuum Workstation Q96 and Q24
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PyroMark Q24 Vacuum Prep Troughs (12)

Cat. No. / ID: 979206

Reusable plastic troughs for use with all PyroMark Q24 Vacuum Workstations
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PyroMark Annealing Buffer (250 ml)

Cat. No. / ID: 979309

For annealing sequencing primer to single-stranded PCR product and for Pyrosequencing reaction
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PyroMark Denaturation Solution (500 ml)

Cat. No. / ID: 979307

For denaturation of double-stranded PCR product into single-stranded template DNA
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PyroMark Q24 Gold Reagents (5 x 24)

Cat. No. / ID: 971802

For 5 x 24 samples for use on the PyroMark Q24 MDx: Enzyme Mixture, Substrate Mixture, and nucleotides
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PyroMark Q24 Cartridge (3)

Cat. No. / ID: 979302

Cartridge for dispensing nucleotides and reagents
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PyroMark Q24 Control Oligo

Cat. No. / ID: 979303

For installation check of PyroMark Q24 MDx system
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PyroMark Binding Buffer (200 ml)

Cat. No. / ID: 979306

For binding of biotinylated PCR product to Sepharose beads
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PyroMark Q24 MDx Vacuum Workstation

Cat. No. / ID: 9001515

Vacuum Workstation (220 V) for preparing 24 samples in parallel, from PCR product to single-stranded template
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PyroMark Q24 Plate (100)

Cat. No. / ID: 979301

24-well sequencing reaction plate for use with PyroMark Q24 MDx; 100 plates in each package
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PyroMark Q24 Validation Oligo

Cat. No. / ID: 979304

For performance check of PyroMark Q24 MDx system
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PyroMark Wash Buffer concentrate (200 ml)

Cat. No. / ID: 979308

For washing of single-stranded DNA

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. PyroMark Q24 MDx Accessories are used in combination with the PyroMark Q24 MDx instrument.

Supporting data and figures

Publications

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
[Quantitative pyrosequencing of heterozygous single nucleotide polymorphisms for rapid diagnosis of Down's syndrome].
Liu XQ; Wu HP; Bu Y; Zou BJ; Chen ZY; Zhou GH;
Zhonghua Yi Xue Yi Chuan Xue Za Zhi; 2009; 26 (3):331-5 2009 Jun PMID:19504451
Association between single nucleotide polymorphisms of surfactant protein D and allergic rhinitis in Chinese patients.
Deng YQ; Tao ZZ; Kong YG; Xiao BK; Chen SM; Xu Y; Wang Y; He Q;
Tissue Antigens; 2009; 73 (6):546-52 2009 Jun PMID:19493231
Global and gene-specific promoter methylation changes are related to anti-B[a]PDE-DNA adduct levels and influence micronuclei levels in polycyclic aromatic hydrocarbon-exposed individuals.
Pavanello S; Bollati V; Pesatori AC; Kapka L; Bolognesi C; Bertazzi PA; Baccarelli A;
Int J Cancer; 2009; 125 (7):1692-7 2009 Oct 1 PMID:19521983
BCL2-938C>A and CALCA-1786T>C polymorphisms in aseptic loosened total hip arthroplasty.
Wedemeyer C; Kauther MD; Hanenkamp S; Nückel H; Bau M; Siffert W; Bachmann HS;
Eur J Med Res; 2009; 14 (6):250-5 2009 Jun 18 PMID:19541585

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
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
How many times can the cartridges for Pyromark Q24 or PyroMark Q96 ID instruments are reused?
When cleaning and storing the cartridges properly the Q24 cartridge can be used up to 30 times and Q96 ID cartridge up to 20 times. The cartridge product sheet and PyroMark Q24/ID user manual contains guidelines how to clean the cartridges correctly.
FAQ ID -2863
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
Which kits can be used in combination with the PyroMark CpG Assays and PyroMark Custom Assays?
QIAamp/DNeasy Kits can be used for DNA isolation, EpiTect Bisulfite Kits for DNA conversion, PyroMark PCR Kit for PCR amplification, EpiTect Control DNA Set for PCR controls, and PyroMark Gold Q24 Reagents or PyroMark Gold Q96 Reagents for the sequencing reaction.

Depending on the platform used, the following reagent kits are required for Pyrosequencing:

PyroMark Q96 ID and MD: PyroMark Gold Q96 Reagents

PyroMark Q24: PyroMark Gold Q24 Reagents

PyroMark Q24 Advanced: PyroMark Q24 Advanced Reagents and PyroMark Q24 Advanced CpG Reagents

PyroMark Q48 Autoprep: PyroMark Q24 Advanced Reagents and PyroMark Q24 Advanced CpG Reagents

FAQ ID -2822
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
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
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
Can PyroMark Gold reagents be vortexed?
Reconstiuted enzyme and substrate of PyroMark Gold Reagents, should not be vortexed since this could lead to conformational changes which affect the activity.
FAQ ID -2844
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