PyroMark PCR Kit

For PCR amplification of template DNA optimized for Pyrosequencing analysis


  • Highly specific amplification of bisulfite converted DNA and gDNA
  • Kit optimized for successful Pyrosequencing analysis
  • High yields of PCR product and no need for optimization
  • Convenient master mix with room-temperature setup
  • Optimized protocols
PyroMark PCR Kit (200)

Cat. No. / ID: 978703

For 200 reactions: 2x PyroMark PCR Master Mix (includes HotStarTaq DNA Polymerase and optimized PyroMark Reaction Buffer containing 3 mM MgCl2 and dNTPs), 10x CoralLoad Concentrate, 5x Q-Solution, 25 mM MgCl2, and RNase-Free Water.
The PyroMark PCR Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

The PyroMark PCR Kit is specifically optimized for Pyrosequencing analysis, providing highly specific and unbiased amplification of template DNA for various applications, such as mutation detection, SNP analysis, methylation analysis, and sequencing. The kit is provided in a convenient master mix format consisting of HotStarTaq DNA Polymerase and optimized PyroMark Reaction Buffer for highly specific amplification. In addition, Q-Solution, a novel additive that enables efficient amplification of "difficult" (e.g., GC-rich) templates, is provided. The PyroMark PCR Kit is also supplied with CoralLoad Concentrate, which is strongly recommended for use with the PyroMark PCR Master Mix, for highly specific PCR and high yields of amplified DNA. CoralLoad Concentrate contains 2 gel-tracking dyes, allowing PCR products to be loaded directly on a gel and checked prior to Pyrosequencing.


The PyroMark PCR Master Mix, with its balanced potassium and sodium salts, promotes specific primer–template annealing and simultaneously reduces nonspecific annealing. Maximum yields of specific products are obtained, even when using extremely low amounts of template (see the figure " Superior results with PyroMark PCR Kit"). This optimized template amplification results in higher Pyrogram peaks, and thus more reliable downstream Pyrosequencing results (see the figure " PCR optimized for Pyrosequencing").

See figures


PyroMark PCR Master Mix

The PyroMark PCR Kit enables PCR amplification of template DNA optimized for Pyrosequencing analysis. The convenient master mix format enables specific amplification of bisulfite converted DNA or genomic DNA from various starting materials, using only one protocol. Bisulfite treatment of genomic DNA converts nonmethylated cytosines to uracils and produces DNA that mainly consists of three bases. This less complex DNA is a challenging PCR template — the PCR products obtained are likely to have low yields, and nonspecific products might also be generated due to increased probability for mispriming. The balanced combination of salts in the PyroMark PCR Kit's unique master mix ensures specifically targeted primer binding and prevents mispriming. The special formulation also prevents accumulation of excess biotinylated primer and minimizes generation of artifacts that can interfere with the Pyrosequencing reaction. The high yields of specific PCR products obtained ensure reliable Pyrosequencing results. 

During the annealing step of every PCR cycle, the buffer composition ensures a high ratio of specific-to-nonspecific primer binding (see figure " Increased specificity of primer annealing"). Owing to a uniquely balanced combination of KCl and (NH4)2SO4, the PCR buffer provides stringent primer annealing conditions over a wider range of annealing temperatures and Mg2+ concentrations, compared with conventional PCR buffers. Optimizing PCR by varying the annealing temperature or Mg2+ concentration is usually not required.

HotStarTaq DNA Polymerase

HotStarTaq DNA Polymerase, provided in the master mix, is a modified form of the recombinant 94 kDa Taq DNA Polymerase from QIAGEN. HotStarTaq DNA Polymerase is provided in an inactive state with no polymerase activity at ambient temperatures. This prevents the formation of misprimed products and primer–dimers at low temperatures. HotStarTaq DNA Polymerase is activated by a 15 minute, 95°C incubation step, that can easily be incorporated into existing thermal cycling programs. HotStarTaq DNA Polymerase provides high PCR specificity and often increases the yield of the specific PCR product. PCR setup is quick and convenient, since all reaction components can be combined at room temperature.


The PyroMark PCR Master Mix is provided with Q-Solution, an innovative PCR additive that facilitates amplification of difficult templates by modifying the melting behavior of DNA. This unique reagent often enables or improves PCR caused by difficult templates (e.g., templates with a high degree of secondary structure or templates that are GC-rich). Unlike other commonly used PCR additives, such as DMSO, Q-Solution is used at just one working concentration, is nontoxic, and PCR purity is guaranteed. PCR fragments amplified in the presence of Q-Solution can be successfully analyzed by Pyrosequencing.

CoralLoad Concentrate

The kit is also supplied with CoralLoad Concentrate, which contains a gel loading reagent and 2 gel tracking dyes (see figure " CoralLoad Concentrate"). When using CoralLoad Concentrate, the PCR products can be directly loaded onto an agarose gel without prior addition of loading buffer. CoralLoad Concentrate can be added to the PCR without affecting amplification sensitivity or specificity. PCR fragments amplified in the presence of CoralLoad Concentrate can be successfully analyzed by Pyrosequencing.


See figures


The reaction composition and cycling conditions are optimized for amplifying template DNA for Pyrosequencing analysis. Template DNA is combined with the convenient PyroMark PCR Mastermix, along with Q-Solution, CoralLoad concentrate, and amplification primers (one of which is biotinylated). HotStarTaq DNA Polymerase is inactive at room temperature, so it is not necessary to keep the reactions on ice during setup.


Pyrosequencing is 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. The PyroMark PCR Kit provides the reagents necessary for reliable and highly specific template amplification for Pyrosequencing analysis.

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
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 Assay Design or application software give any cycling conditions for individual assay primers or a PCR setup pipetting scheme?
The PyroMark Assay Design and application software do not support PCR setup with a pipetting scheme or PCR cycling conditions. General recommendations how to setup and optimization of the PCR reaction are contained in the PyroMark PCR handbook.
FAQ ID -2862
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
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
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