PyroMark Q96 Tests

For analyzing gene-specific sequence variation or methylation on PyroMark Q96 ID or PyroMark Q96 MD


  • Detection and quantification of mutations in cancer-related genes
  • Quantification of individual or multiple CpG sites in different genes
  • Estimation of global CpG methylation in defined regions of interest
  • Sequence context provides built-in quality control of the assays
PyroMark HFE (96)

Cat. No. / ID: 972442

For detection of variants in the HFE gene (2 x 96 reactions on PyroMark Q96 MD, 1 x 96 reactions on PyroMark Q96 ID, 8 x 24 reactions on PyroMark Q24)
CpG Prader-Willi
CpG p16
PyroMark APOE, PyroMark HFE, PyroMark MTHFR, PyroMark Q96 CpG LINE-1, PyroMark Q96 CpG MGMT, PyroMark Q96 CpG MLH1, PyroMark Q96 CpG p16, and PyroMark Q96 CpG Prader-Willi tests are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

PyroMark Q96 Tests are a range of predesigned assays suitable for highly reliable, quantitative analysis of mutations or SNPs, as well as CpG methylation. Tests designated specifically with 'Q96' are optimized for use with the PyroMark Q96 MD and/or PyroMark Q96 ID Instruments. Other tests, such as PyroMark HFE and PyroMark MTHFR, can additionally be performed on the PyroMark Q24 (with the exception of PyroMark APOE).


The PyroMark Q96 Tests are highly suitable for accurately measuring the CpG methylation status, as exemplified with the PyroMark Q96 CpG p16 Test (see figure  PyroMark p16 analysis).
See figures


PyroMark Q96 Tests used in combination with PyroMark Q96 MD and/or PyroMark Q96 ID Systems enable accurate, quantitative mutation (or SNP) and methylation analysis by Pyrosequencing. The assays consist of PCR primers and a sequencing primer.


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 Q96 Tests are predesigned assays for methylation analysis by Pyrosequencing. Each test includes the primers necessary for generating the biotinylated PCR product, as well as the primer for the Pyrosequencing reaction.


PyroMark Q96 Tests enable highly accurate, quantitative methylation and mutation (or SNP) analysis applicable in several fields of research, including cancer and epigenetics.

Supporting data and figures


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
Does the PyroMark CpG LINE assay target mouse transposons as well?

The PyroMark CpG LINE-1 assay is specific for human DNA and was not tested on mouse DNA.  Mouse LINE-1 elements differ slightly in sequence and may not be detected.



Can the PyroMark Q96 CpG LINE assay be used with an ID system?
The PyroMark Q96 CpG LINE-1 assay can only be used with a PyroMark Q96 MD system because the PyroMark Q96 ID instrument does not have a sensitive enough camera. For the Pyromark Q24 there is a dedicated PyroMark Q24 LINE-1 assay.
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 amplicon length of the PyroMark CpG LINE assay?
The amplicon length of the PyroMark CpG LINE-1 PCR product is about 146 bp. for PyroMark Q24 and PyroMark Q96 MD.
Does the PyroMark LINE assay target all LINE sequences?
The PyroMark assay is designed to cover LINE-1 retrotransposable elements in the genome. Those differ slightly in sequence and it might be that not all are picked up by our LINE-1 assay. The intention of the assay is to cover as many as possible of the LINE-1 sequences.
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 region of LINE gets targeted by the assay for PyroMark Q96 MD or PyroMark Q24?

The PyroMark Q96 CpG LINE-1 for PyroMark Q96 MD is designed to target four CpG sites in positions 331 to 305 (GenBank accession no X58075). The PyroMark Q24 CpG LINE-1 assay for PyroMark Q24 targets three CpG sites in positions 331 to 318 (GenBank accession no X58075). The target region is human LINE-1 transposon DNA consensus sequence.

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
How many CpG sites are analyzed by the PyroMark CpG LINE assay?
The PyroMark Q24 LINE-1 assay covers 3 CpG sites and the LINE-1 assay for PyroMark Q96 MD covers 4 sites.
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
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