For quantitative analysis of genetic or epigenetic DNA modifications using Pyrosequencing technology
For quantitative analysis of genetic or epigenetic DNA modifications using Pyrosequencing technology
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.
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.
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.
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.
|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|
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.
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").
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.
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.
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.
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.
PyroMark Assay Design Software 2.0 ensures easy design of PCR and sequencing primers. The assays are optimized for use with all PyroMark instruments.
|Instrument dimensions||420 x 390 x 525 mm (16.5 x 15.4 x 20.7 in)|
|Connections||One USB port (2.0)|
|Applications||Methylation analysis, allele quantification, genotyping, sequence analysis|
|Humidity||Relative humidity of 20–90% (noncondensing)|
|Kits designed for this instrument||PyroMark Q24 Tests|
|Operating temperature||15–32°C (59–90°F)|
|Place of operation||For indoor use only|
|Chemical resistance||pH 4 to pH 9, common detergents, 0.5 M sodium hydroxide, ethanol|
|Power||100–240 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 temperature||28°C (82.4°F) ± 1%|
|Process time||Depends on the number of dispensations (20 dispensations take 24 minutes)|
|Samples per run (throughput)||1–24|
|Software||PyroMark Q24 Software 2.0 (to be installed on PC)|
|Weight||27.5 kg (60.6 lb)|
|Altitude||Up to 2000 m (6500 ft)|
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.
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.
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.
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.
Please install software 2.0.7, which is compatible with Windows 7, 64 and 32 bit.
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.
The estimated dispensation time is 1 min/nucleotide.
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.