Pyromark id user manual

PyroMark Q24 facilitates accurate and highly sensitive mutational analysis of any gene of interest, and enables quantification of allele representation in mixed cell populations. 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. 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.

Apyrase, a nucleotide-degrading enzyme, continuously degrades unincorporated nucleotides and ATP. When degradation is complete, another nucleotide is added. Step 5: Addition of dNTPs is performed sequentially. 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 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. The assays are optimized for use with all PyroMark instruments. An instrument method or instrument code encodes the individual pulse time settings of specific cartridge lot batch. These pulse time settings change when e. 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. 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.

This acts as a useful quality control for full conversion of unmethylated C residues during bisulfite treatment and PCR. PyroMark instruments offer a range of throughput scales. 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 minutes. 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.

Usually the sequencing primer is used at 0. The PyroMark Q48 Autoprep dispenses the sequencing primers for annealing. The final concentration of sequencing primers in a well is 0. PyroMark Q24 analyses up to 24 individual samples in a single run. The time needed to perform a run depends on the number of nucleotides dispensed in the specific assay. Typical reading length using Pyrosequencing technology is 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 or more bases can be obtained in just a single reaction with the Q48 PyroMark Autoprep.

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. Reorder now! Reorder from your past orders in just one click. Order by Quote. Quote Number. Add quote number from your quote document.

Customer Number. Add customer number from your quote document. To remove a quote go to the Cart. View Quote Example. Catalog Number. Looking for a quick way to design experiments? 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 or more bases can be obtained in just a single reaction with the Q48 PyroMark Autoprep. Reorder now! Reorder from your past orders in just one click. Order by Quote. Quote Number. Add quote number from your quote document. Customer Number.

Add customer number from your quote document. To remove a quote go to the Cart. View Quote Example. Catalog Number. Looking for a quick way to design experiments? Try the Workflow Configurator. A convenient tool to build experimental workflows and find products to match your needs. Log Out. Show More. Software Product. PyroMark Software. Pyro Q48, Preventive Subscription. PyroMark Q96 ID is a powerful sequencing and quantification platform highly suited for epigenetics, mutation gene expression analysis, and microbial identification and resistance typing.

With its well format, automatic base-calling function, and dedicated software solutions for methylation analysis and assay design, the PyroMark Q96 ID can handle any research question requiring true sequence information and quantification of genetic or epigenetic variation.

Linearity of methylation quantification. Analysis of a tri-allelic SNP. Principle of Pyrosequencing — step 2. The first deoxribonucleotide triphosphate dNTP is added to the reaction. Each incorporation event is accompanied by release of pyrophosphate PPi , in a quantity equimolar to the amount of incorporated nucleotide.

Principle of Pyrosequencing — step 3. The height of each peak light signal is proportional to the number of nucleotides incorporated. Principle of Pyrosequencing — step 4. Principle of Pyrosequencing — step 5. Steps of the Pyrosequencing reaction: Step 1: A DNA segment is amplified, and the strand to serve as the Pyrosequencing template is biotinylated.

Principle of Pyrosequencing — step 1. Fast and easy sample preparation From PCR product to single-stranded template ready for sequencing — up to 96 samples can be prepared in parallel using the PyroMark Q96 Vacuum Workstation in less than 15 minutes.

Supporting data and figures Analysis of antibacterial resistance in Helicobacter pylori. Analysis of mutations in the 23S genes that confer antibacterial resistance in Helicobacter pylori. Analysis of antibacterial resistance in Helicobacter pylori. Fully integrated system. Simply design the necessary assay and run files, load your samples and reagents, and walk away. PyroMark Q96 ID. The right instrument for your needs.

By processing 96 samples in a single run, the PyroMark Q96 ID combines analysis versatility with high throughput. The PyroMark Q96 MD explands this throughput with a robotic module that enables hands-free processing of ten well plates. New assay design can be done with the PyroMark Q24, which offers the same analysis versatility on a smaller throughput scale.

Intuitive software. Efficient template prep. Workflow solutions. The components of the PyroMark Q96 ID System are designed to make your research workflow straightforward and efficient. Each step is supported by software, kits, reagents, and sample preparation instrumentation that are optimized for Pyrosequencing. Resources White Papers 1. EN - Techniques to overcome bottlenecks in epigenetics research EN.

PDF 1MB. Download Files ZIP KB. Safety Data Sheets 1. Safety Data Sheets EN. Instrument User Manuals 2. PDF 4MB. Pyrosequencing — the synergy of sequencing and quantification EN. PDF 2MB. Kit Handbooks 1. PDF KB. Operating Software 1. The main feature of this release is 64bit compatibility with Windows 7 operating systems.

Publications Genetic diagnostics of functional variants of the human dopamine D2 receptor gene. A pyrosequencing assay for the rapid discrimination of mitochondrial lineages in the Salmo trutta species complex. Use of Pyromark Q96 ID pyrosequencing system in identifying bacterial pathogen directly with urine specimens for diagnosis of urinary tract infections.

PyroMark plate. Position the plate on the workstation. Fill the workstation troughs according to the diagram to the right. Start the pump and apply vacuum to the tool by opening the switch. Flush the filter probes with high-purity water Milli-Q. Refill the trough with fresh. Position the PCR plate on the workstation. Ensure that both plates are.

Check the lot. For lot number For lot numbers lower than. This is not a complete list of suppliers and does. Preparing the master mix to immobilize the PCR product.