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PyroMark Custom Assays

For Pyrosequencing analysis of any user-defined DNA sequence


  • Ready-to-use assays produced from user-designed primer sequences
  • Convenient ordering through GeneGlobe
  • Delivers optimized primer concentration and quality
  • Supported by QIAGEN's expertise in Pyrosequencing analysis
Configure at GeneGlobe
Find or custom design the right target-specific assays and panels to research your biological targets of interest.
PyroMark Custom Assay (200)

Cat. No. / ID: 978776

User-designed PCR and sequencing primers for Pyrosequencing analysis (200 reactions; tube format)
Configure at GeneGlobe   to see pricing.
PyroMark Custom Assays are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.
Configure at GeneGlobe
Find or custom design the right target-specific assays and panels to research your biological targets of interest.

Product Details

PyroMark Custom Assays deliver ready-to-use assays based on user-designed primers for the Pyrosequencing analysis of any DNA sequence.


PyroMark Custom Assays give users the flexibility to order a high-quality Pyrosequencing kit for the analysis of any DNA sequence. Enter the sequence of pre-designed PCR and sequencing primers, and receive a ready-to-use assay with optimized primer concentrations. The source of the primer sequences you enter may be from the literature, from previous projects, or — to maximize analysis success — from primer design and evaluation using the PyroMark Assay Design Software 2.0. Combined with other dedicated products of the PyroMark line, including carefully formulated PCR and sequencing reagents, the PyroMark Custom Assays expand the streamlined Pyrosequencing workflow to enable analysis of virtually any sequence in any genome.

Optimized primer concentration

Both the PCR and sequencing primers included in PyroMark Custom Assays are delivered at optimal concentrations for convenience of use and enhanced PCR quality. Together with the carefully formulated reagents of the PyroMark PCR Kit, this optimization ensures improved yield and quality of sequencing template, which consequently increases assay reliability. If working with RNA samples, the new PyroMark OneStep RT-PCR Kit enables reverse transcription and template amplification in one reaction with the same optimized yield and assay reliability.

The advantages of GeneGlobe

QIAGEN's GeneGlobe Web portal is a one-stop online interface that grants access to our extensive range of assay technologies in a gene- or pathway-specific context. GeneGlobe brings together state-of-the-art search and design algorithms, up-to-date sequence data, and our innovative technologies to facilitate discovery. Simply enter a search term for the gene or pathway of interest, and let GeneGlobe show you how to advance your research in epigenetics, gene expression, genetic determinants of disease, and more (see figure " Ordering through GeneGlobe").

See figures


Prior to Pyrosequencing, a biotinylated PCR product is generated. Biotinylated PCR products are bound to streptavidin-coated Sepharose beads. The beads are then captured with the vacuum tool on the PyroMark Vacuum Workstation, and throroughly washed, generating single-stranded DNA suitable for sequencing. PyroMark Custom Assays give users the flexibility to order a high-quality custom-designed Pyrosequencing kit for the analysis of any DNA sequence. Each assay includes the primers necessary for generating the biotinylated PCR product, as well as the primer for the Pyrosequencing reaction.


Pyrosequencing is becoming increasingly important for research applications in a variety of disciplines. Pyrosequencing enables powerful and versatile analysis of genetic and epigenetic variation, 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. PyroMark Custom Assays provide the high-quality ready-to-use primers for such analyses.

Supporting data and figures


Safety Data Sheets (1)
Download Safety Data Sheets for QIAGEN product components.


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 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
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 is included in a PyroMark Custom Assay?
The Pyromark Custom Assay includes a 10x PCR Primer Set (mixture of forward and reverse PCR Primer) and a 10x Sequencing Primer. Reagents for performing PCR and Pyrosequencing reaction are not included.
FAQ ID -2815
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
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
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
Does QIAGEN offer a design of Custom PyroMark CpG Assays?
No, QIAGEN does not design any Custom PyroMark CpG Assay. Customers have the possibility to order pre-designed, genome-wide PyroMark CpG Assays or order a user-designed assay (e.g. with the PyroMark Assay Design Software or assays known from previous projects or from the literature).
FAQ ID -2818
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
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 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 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