PyroMark Q24

For quantitative analysis of genetic or epigenetic DNA modifications using Pyrosequencing technology

Features

  • Assay versatility on the same instrument and in the same run
  • Reliable quantification of allele representation and methylation status
  • Sequence information enables discovery of rare mutations
  • 1–24 samples can be analyzed in as little as 15 minutes
  • Compact detection platform uses minimal bench space

Product Details

The PyroMark Q24 uses Pyrosequencing technology for real-time, sequence-based detection and quantification of sequence variants and epigenetic methylation. The PyroMark Q24 is highly suited for the analysis of CpG methylation, SNPs, insertion/deletions, STRs, variable gene copy number, as well as for microbial identification and resistance typing.

Explore the virtual demo to learn more about the PyroMark Q24.

Performance

A detection tool highly suited for epigenetics research

Pyrosequencing complements QIAGEN's epigenetics portfolio and enables accurate and sensitive quantification of methylation levels by providing highly reliable sequence data (see figure " CpG methylation analysis of the MLH1 gene"). It even allows the identification of novel mutations, as well as detection of aberrant DNA methylation patterns present at low levels. PyroMark Q24 includes a complete software package for CpG methylation analysis and a built-in control for bisulfite treatment.

Quantification of individual CpG sites

Analyzing individual CpG sites is crucial when studying differential gene expression in various tumors (see figure " CpG methylation pattern in the RASSF1A gene"). The lower resolution data provided by traditional methods fail to provide this degree of sensitivity. Pyrosequencing technology overcomes this challenge and enables analysis of single variations in the methylation pattern of single or multiple CpG sites with high accuracy.

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 is a fully integrated system that provides real-time sequence information, and is highly suitable for epigenetic research and genetic analysis. The system includes PyroMark Q24, PyroMark Q24 Vacuum Workstation, PyroMark Q24 Software, PyroMark Gold Q24 Reagents, PyroMark Control Oligo, and PyroMark Q24 Validation Oligo (see table). Sample preparation solutions are also supplied to enable preparation of single-stranded DNA using the PyroMark Q24 Vacuum Workstation.

System component Description
PyroMark Q24 Sequencing instrument for quantitative mutational and methylation analysis
PyroMark Q24 Vacuum Workstation Workstation for sample preparation of up to 24 samples in parallel
PyroMark Q24 Software Analysis software; provided in 2 analysis modes (for CpG analysis and allele quantification)
PyroMark Q24 Gold Q24 Reagents Enzymes, substrates, and nucleotides
PyroMark Q24 Control Oligo Control for verification of proper installation and operation of the system
PyroMark Q24 Validation Oligo Control for performance confirmation of the system
PyroMark Q24 system.
A highly suitable platform for genetic analysis

Genetic analysis comprises multiple applications to analyze differences in genomic DNA, including mutation detection and SNP typing. PyroMark Q24 facilitates accurate and highly sensitive mutational analysis of any gene of interest, and enables quantification of allele representation in mixed cell populations. QIAGEN also offers optimized and validated RUO tests for analyzing particular gene mutations by Pyrosequencing.

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 alpha-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").

Streamlined workflow — from sample to result

The versatile PyroMark Q24 seamlessly integrates into epigenetics and genetic analysis workflows, and complements QIAGEN's advanced technologies for sample preparation, bisulfite conversion, and PCR amplification. The highly reliable instrument enables sequence-based detection and quantification of CpG sites as well mutations. The streamlined workflow means that results can be achieved faster.

See figures

Procedure

Fast and easy sample preparation 

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

Applications

Pyrosequencing is becoming increasingly important for research applications in a variety of disciplines. 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 Q24 enables powerful and versatile analysis of genetic and epigenetic variation. In addition, because Pyrosequencing integrates sequence detection and quantification, the enhanced analysis resolution can lead to new discoveries.

Software

Easy-to-use PyroMark Q24 software 

PyroMark Q24 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.

Flexible and simple Pyrosequencing assay design using PyroMark Assay Design Software

PyroMark Assay Design Software 2.0 ensures easy design of PCR and sequencing primers. The assays are optimized for use with all PyroMark instruments.

Supporting data and figures

Specifications

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

Resources

Operating Software (1)
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 software license and registered PyroMark Q24 instrument. If you do not have a valid software license, contact your QIAGEN sales representative.
Application Notes (3)
Download Files (15)
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0001
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0002
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0003
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0004
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0005
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0006
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0007
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0008
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0009
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0010
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0011
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0012
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0013
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0014
Instrument methods file for use with PyroMark Q24 Software, PyroMark Gold Q24 Reagents, and PyroMark Q24 Cartridges marked with method code 0015
Instrument User Manuals (1)
Software User Guides (1)
Scientific Posters (1)

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
Assessing combined methylation-sensitive high resolution melting and pyrosequencing for the analysis of heterogeneous DNA methylation.
Candiloro IL; Mikeska T; Dobrovic A;
Epigenetics; 2011; 6 (4):500-7 2011 Apr 1 PMID:21364322
Cell specific patterns of methylation in the human placenta.
Grigoriu A; Ferreira JC; Choufani S; Baczyk D; Kingdom J; Weksberg R;
Epigenetics; 2011; 6 (3):368-79 2011 Mar 1 PMID:21131778
CpG island hypermethylation of the neurofibromatosis type 2 (NF2) gene is rare in sporadic vestibular schwannomas.
Kullar PJ; Pearson DM; Malley DS; Collins VP; Ichimura K;
Neuropathol Appl Neurobiol; 2010; 36 (6):505-14 2010 Oct PMID:20831745
Systematic cross-validation of 454 sequencing and pyrosequencing for the exact quantification of DNA methylation patterns with single CpG resolution.
Potapova A; Albat C; Hasemeier B; Haeussler K; Lamprecht S; Suerbaum S; Kreipe H; Lehmann U;
BMC Biotechnol; 2011; 11 :6 2011 Jan 14 PMID:21235780

FAQ

What are the differences between new PyroMark Q24 Advanced reagents chemistry and PyroMark Q24 Gold reagents?

The PyroMark Q24 Advanced reagent kit contains an improved enzyme and substrate mix and a new annealing buffer for the new adapted annealing procedure (adapted reaction volume of 20 µl and adapted annealing protocol). Moreover, the kit also contains binding buffer and the dATPalpha is half concentrated.

FAQ ID -3165
Which operating system is compatible with PyroMark IdentiFire Software?

PyroMark IdentiFire Software is compatible with Windows 2000, Windows XP, and Windows 7 (32 bit).

 

FAQ ID - 3340
In which format can PyroMark CpG Assays be ordered?
PyroMark CpG Assay can be ordered in tubes or on 96-well plates. PyroMark CpG Assays, 96 wells, require a minimum order of 24 assays per plate.
FAQ ID -2824
Is there a user manual available for the PyroMark Assay design software?
There is no specific PyroMark Assay Design Software user manual available but a so-called Quick Guide can be downloaded from the PyroMark instrument webpage. Furthermore, the software contains a comprehensive online help (accessible via the Help menu or by pressing the “F1” key).
FAQ ID -2851
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 cat. 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 cat. 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
How much space does the PyroMark Q24 instrument need?

PyroMark Q24 takes very little bench space, measuring only H420 x W390  x D525 mm.

 

FAQ ID -2098
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, for example, 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
How does the built-in QC control for complete bisulfite conversion of DNA work on PyroMark Q24?

Data generated with the methylation analysis software (detection and quantification of CpG sites) on PyroMark Q24 contains unique features that act as quality control for complete bisulfite conversion of DNA. When the assay encounters a C not followed by a G (C unmethylated), that C should be fully converted to T if the bisulfite treatment upfront was successful. Subsequently, it should be presented in the pyrogram as T = 100%. This acts as a useful quality control for full conversion of unmethylated C residues during bisulfite treatment and PCR.

FAQ ID -2095
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 on how to setup and optimization of the PCR reaction are contained in the PyroMark PCR Handbook.
FAQ ID -2862
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.
FAQ ID -2839
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 2 mm orbital diameter.
FAQ ID -2837
How many times can the cartridges for PyroMark Q24 or PyroMark Q96 ID instruments be 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 contain 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
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 camera that is sensitive enough. For the PyroMark Q24, there is a dedicated PyroMark Q24 LINE-1 assay.
2861
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 10x Sequencing Primer. Reagents for performing PCR and pyrosequencing reaction are not included.
FAQ ID -2815
What are the features of PyroMark CpG Assays, for example, in terms of design and validation?
PyroMark CpG Assays are genome-wide, pre-designed methylation assays for pyrosequencing analysis. An optimized design algorithm was used for highly specific assay design and advanced CpG methalytion results.
FAQ ID -2821
What is the sensitivity limitation for pyrosequencing?
In general, the standard claim for pyrosequencing sensitivity is approximately 5%, which is also published in many papers. The actual sensitivity limit is assay dependent and has to be determined individually.
FAQ ID -2840
How do I perform the PyroMark Q24 Advanced upgrade?

The upgrade can be performed by the user since it only requires installation of the application software and download and installation of the new firmware according to instruction in the Q24 user manual. A service engineer will not be required onsite for the upgrade.

FAQ ID -3163
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:

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
Is it possible to use PyroMark Q24 Advanced reagents on PyroMark Q24 system?

PyroMark Q24 Advanced reagents should only be used in combination with PyroMark Advanced system. Usage of these reagents on PyroMark Q24 system will result in decreased pyrosequencing performance and low signals.

FAQ ID -3160
We recently changed the OS from Windows XP to Windows 7. When re-installing software Pyromark Q24 2.0.6, it fails to analyze the results. Any suggestions?

Please install software 2.0.7, which is compatible with Windows 7, 64 and 32 bit.

FAQ ID - 3621
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 that are extended or the primers itself form dimmers that 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 sample throughput of pyrosequencing systems?

PyroMark instruments offer a range of throughput scales. The PyroMark Q24 can process 1–24 samples in parallel, the PyroMark Q48 Autoprep, 1–48; the PyroMark Q96 ID, 1–96; and the PyroMark Q96 MD, 1–96; or the automation option enables automated processing of ten 96-well plates. The sample processing speed depends on the number of nucleotide dispensations necessary for the programmed analysis. Twenty dispensations take approximately 24 minutes on all instruments; thus, 96 samples are typically processed in 10–100 minutes.

 

 

FAQ ID -2215
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.
2856
What is the concentration of PyroMark Control Oligo?
PyroMark Control Oligo has a concentration of 20 µM and is delivered in a volume of 50 µl. Two tubes of 10x dilution buffer (2x 1.7 ml) are delivered with the control oligo.
FAQ ID -2846
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.
2860
How are the PyroMark CpG Assays shipped and stored?
PyroMark CpG Assays are shipped lyophilized at ambient temperatures (20−25°C) and should be stored at −20°C either reconstituted or lyophilized. Repeated freeze−thaw cycles should be avoided. When stored under these conditions, the reconstituted product can be kept for at least 18 months from the date of receipt without reduction in performance.
FAQ ID -2816
How are the PyroMark CpG Assays reconstituted?
The PyroMark CpG Assay is reconstituted as a 10x PCR Primer Set in 550 µl TE, pH 8.0 and the 10x Sequencing Primer is reconstituted in 1175 µl Annealing Buffer if using the PyroMark Q24, 880 µl if using the PyroMark Q96 ID, and 1175 µl if using the PyroMark Q96 MD.
FAQ ID -2817
How many times can vacuum troughs be reused 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 reused. 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 50 mm x 60 mm and for PyroMark Q96 ID/MD 120 mm x 80 mm.
FAQ ID -2838
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.

2857
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 0.3 µM 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 0.3 µM and, for PyroMark Q96 ID, 0.4 µM.

The PyroMark Q48 Autoprep dispenses the sequencing primers for annealing. The final concentration of sequencing primers in a well is 0.8 µM 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
Where can I find explanations to the warning given by the PyroMark software after run data analysis?
The PyroMark Q24 application software a PyroMark Q24 Software user guide with all essential information about warnings and software features, which can be found contains under Help (press F1). The Pyromark ID/MD software also contains a software guide under Help. Moreover, the individual instrument user manuals contain helpful information in the troubleshooting section.
FAQ ID -2874
Will the primer sequence for the PyroMark CpG Assay be provided?
Primer sequences for PyroMark CpG Assays are not provided; they are proprietary.
FAQ ID -2823
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.
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 might 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 to reduce contamination risk with PCR amplicons from previous PCRs.
FAQ ID -2843
Is the CpG software included in the PyroMark instruments to study methylation status?
The PyroMark Q24 software and the new PyroMark Q96 ID software version 2.5 support CpG analysis in the CpG mode. The ORACLE-based PyroMark Q96 ID software version and PyroMark Q96 MD software do not support CpG analysis. In this case, an independent, additional software is needed which is the PyroMark CpG software version 1.0.
FAQ ID -2842
How long does a single run on the PyroMark Q24 take?

The estimated dispensation time is 1 min/nucleotide.

FAQ ID -2096
How many CpG sites are analyzed by the PyroMark CpG LINE assay?
The PyroMark Q24 LINE-1 assay covers three CpG sites, and the LINE-1 assay for PyroMark Q96 MD covers four sites.
2858
What is the purpose of the unmethylated and unconverted control DNA of the EpiTect PCR Control DNA Set?

The unmethylated and unconverted human control DNA of the EpiTect PCR Control DNA Set allows to check that primers designed for the specific detection of unmethylated and converted DNA (U-converted DNA), and for methylated, converted DNA (M-converted DNA) does not bind to untreated genomic DNA.*

In case bisulfite conversion was not complete, leaving certain unmethylated C residues unconverted, false positives would result if the primer specific for M-converted DNA binds to untreated gDNA.

This control DNA can also be used to check conversion efficiency during bisulfite treatment.

 

*Summary of principle: Methylation of DNA occurs on cytosine residues, especially on CpG dinucleotides enriched in small regions of DNA. Incubation of target DNA with sodium bisulfite, using, for example, EpiTect Bisulfite Kits, results in conversion of unmethylated cytosine residues into uracil, leaving methylated cytosines unchanged.

 

FAQ ID -2007
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 bases or more can be obtained in just a single reaction with the Q48 PyroMark Autoprep.

 

 

FAQ ID -2216
What is the recommended amplicon size for CpG assays?
The amplicon length should be short (<200 bp). This is critical especially for DNA from FFPE tissue that 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 is possible to use so far unused pyrosequencing wells for the next run and leave the wells that are already used empty. However, due to contamination risk when cleaning and handling plates, QIAGEN does not recommend this.
FAQ ID -2872
How do I set up a PyroMark CpG Assay?
All relevant information regarding PyroMark CpG Assay setup can be found on the GeneGlobe website. For the Q24 and Q96, the "Sequence to Analyze" and dispensation order should not be copied manually to create a new assay. Instead, the assay file should be downloaded from the web and opened in the PyroMark CpG softwarePyroMark Q96  ID v2.5 (or higher) software, and PyroMark Q24 Software to keep important software settings.

When using the PyroMark CpG assays with the PyroMark Q48, use the "Sequence to Analyze" provided in the "product specification" section to create an assay setup file in the PyroMark Q48 software. This is done by selecting New CpG assay and pasting in the Sequence to Analyze (not the "sequence after bisulfite treatment") into the Sequence Before Bisulfite Treatment field and pressing Create Dispensation order.
FAQ ID -2814
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? If this method cannot be selected automatically in the application software, you can download the method/code file from the instrument webpage.
Let the PyroMark instrument warm up (approx. 60 min) to adapt to room temperature before use. Make sure the ambient room temperature is within range 18−28°C.
FAQ ID -2878