PyroMark Q96 ID - High-Resolution Genetic Analysis In 96-Well Format

For quantitative methylation analysis, sequence variation analysis, and microbial typing using Pyrosequencing technology

• Sequence context provides built-in quality control of the assay
• Enables a broad range of analyses, powered by intuitive software
• Analysis of one assay with up to 96 individual samples or up to 96 different assays in one plate

The PyroMark Q96 ID System includes the PyroMark Q96 ID Instrument, the PyroMark Q96 Vacuum Workstation for sample preparation, PyroMark Q96 ID Software and PyroMark supplementary software for versatile assay setup and results analysis, optimized reagents, and dedicated PyroMark Test Kits (see Related Products).

Pyrosequencing technology, which is based on the principle of sequencing by synthesis, provides quantitative data in sequence context within minutes. PyroMark Q96 ID is a fully integrated system that provides real-time sequence information and is highly suitable for detection of genetic variations, genetic quantification, and short DNA sequencing. The following products are used in combination with PyroMark Q96 ID instrument - PyroMark Q96 Vacuum Workstation, PyroMark CpG SW, PyroMark Assay Design SW, PyroMark IdentiFire SW, PyroMark Gold Q96 Reagents, and PyroMark Control Oligo. Sample preparation solutions are also supplied to enable preparation of single-stranded DNA using the PyroMark Q96 Vacuum Workstation.

The Pyrosequencing reaction involves five steps:

Step 1 A sequencing primer is hybridized to a single-stranded PCR amplicon that serves as a template, and incubated with the enzymes, DNA polymerase, ATP sulfurylase, luciferase, and apyrase as well as the substrates, adenosine 5` phosphosulfate (APS), and luciferin.

Step 2

The first deoxribonucleotide triphosphate (dNTP) is added to the reaction. DNA polymerase catalyzes the incorporation of the deoxyribo-nucleotide triphosphate into the DNA strand, 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.

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 a charge coupled device (CCD) chip 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.

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. It should be noted that deoxyadenosine alfa-thio triphosphate (dATP•S) 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 built up and the nucleotide sequence is determined from the signal peaks in the Pyrogram trace.

Streamlined workflow - from sample to result
The versatile PyroMark Q96 ID 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 quantification and detection of SNPs, insertion- and deletion- mutations, CpG sites, as well as generation of sequence information. The streamlined workflow means that results can be achieved faster.

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 all PyroMark instruments.

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. The workstation ensures easy handling and the actual hands-on time is less than 5 minutes.

Easy-to-use PyroMark Q96 ID software
PyroMark Q96 ID software enables comprehensive analysis of your results. The software contains two analysis modes - SQA and SNP. Both of these modes can be used to analyze samples on the same plate, enabling different types of samples to be run at the same time. The SNP mode can be used for analysis of single and multivariable positions as well as di-, tri- , and tetra- allelic mutations. The SQA mode enables sequence identification.

Quantitative analysis of methylation in single and multiple CpG sites with PyroMark CpG SW 1.0.
As a complement to PyroMark Q96 ID software, PyroMark CpG SW 1.0 provides a complete solution for a range of analyses, from estimations of global methylation to quantification of single and multiple consecutive CpGs.

Integrated sequencing, identification and resistance typing for clinical microbiology
The real-time sequencing capability of Pyrosequencing technology enables microbial identification in a single assay. PyroMark Q96 ID analyzes the full genetic diversity of resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may be involved in the resistance pattern.

Using PyroMark Q96 ID for microbial identification
Pyrosequencing technology provides sequence and identity information of a broad range of organisms with high sensitivity and in less than 1 hour. Compared to traditional sequencing methods, these advantages make Pyrosequencing technology the ideal choice for microbial analysis. PyroMark Q96 ID together with PyroMark IdentiFire SW enables convenience and results in substantial time-savings.

A detection tool highly suited for epigenetics research
Pyrosequencing complements QIAGEN`s epigenetics portfolio and enables accurate and sensitive quantification of the methylation status by providing highly reliable sequence data. Traditional methods often fail to provide this degree of sensitivity since the data obtained is of lower resolution. PyroMark Q96 ID together with PyroMark CpG SW 1.0 is a complete package for estimation of global methylation and for analysis and quantification of single and multiple consecutive CpGs. In addition, the system provides the user with a built-in control for bisulfite treatment.

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 Q96 ID facilitates accurate and highly sensitive mutational analysis of any gene of interest and enables quantification of allele representation in mixed cell populations. Pyrosequencing technology offers RUO tests for mutation analysis of genes such as KRAS.

An invaluable tool in a variety of research areas
Pyrosequencing is becoming increasingly relevant 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 Q96 ID enables powerful and versatile analysis of genetic and epigenetic variation. In addition, because Pyrosequencing integrates sequence detection and quantification, the heightened analysis resolution can lead to new discoveries.

• Instrument dimensions: 490 x 540 x 620 mm (19.3 x 20.5 x 24.4 in)
• Weight: 52 kg (114.6 lb)
• Applications: Methylation analysis, allele quantification (SNP, InDels), sequence analysis
• Samples per run; throughput: 1–96
• Technology: Pyrosequencing
• Kits designed for this instrument: PyroMark Q96 Tests
• Process time: 10 to 100 minutes for up to 96 samples in parallel
• Process temperature: 28°C (82.4°F) ± 1%
• Software: PyroMark Q96 Software (included), PyroMark CpG SW 1.0 (supplementary), PyroMark IndentiFire SW 1.0 (supplementary), PyroMark Assay Design SW 2.0 (supplementary)
• Power: 100–120 V AC, 220–240 V AC; 56–60 Hz
• Operating temperature: 18–28°C (64–82°F)
• Altitude: Up to 2000 m (6500 ft)
• Place of operation: For indoor use only
• Pollution level: 2
• Overvoltage category: II