PyroMark Q96 ID

For quantitative analysis of genetic or epigenetic DNA modifications using Pyrosequencing technology
  • Sequence context provides built-in quality control of the assay
  • Enables a broad range of analyses, powered by intuitive software
  • Run up to 96 different assays or 96 samples with one assay together
  • Fast delivery of direct and unambiguous sequence data
 
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 96-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.
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产品 货号 目录价:
PyroMark Q96 ID System
Instrument and software for Pyrosequencing analysis: includes installation, training, and 1-year warranty on parts and labor
9001525
询价
PyroMark Q96 ID Priority Package
Instrument and software for Pyrosequencing analysis: includes Priority Package with installation, training, 2-year warranty on parts and labor, and 2 preventive maintenance visits
9001915
询价
PyroMark Q96 ID Priority Package Plus
Instrument and software for Pyrosequencing analysis: includes Priority Package with installation, training, 3-year warranty on parts and labor, and 3 preventive maintenance visits
9001916
询价
PyroMark Q96 ID Software 2.5
Application software for PyroMark Q96 ID systems
9022198
询价

The PyroMark Q96 ID Instrument and PyroMark Q96 Vacuum Workstation are intended to be used only in combination with QIAGEN kits indicated for use with the PyroMark Q96 ID for the applications described in the kit handbooks. If the PyroMark Q96 ID Instrument and PyroMark Q96 Vacuum Workstation are used with other than QIAGEN kits, it is the user's responsibility to validate the performance of such product combination for any particular application.
Analysis of antibacterial resistance in Helicobacter pylori.|Analysis of multiple contiguous CpG sites.|Analysis of a tri-allelic SNP.|Principle of Pyrosequencing — step 1.|Principle of Pyrosequencing — step 2.|Principle of Pyrosequencing — step 3.|Principle of Pyrosequencing — step 4.|Principle of Pyrosequencing — step 5.|Linearity of methylation quantification.|Fully integrated system.|PyroMark Q96 ID.|The right instrument for your needs.|Intuitive software.|Efficient template prep.|Workflow solutions.|
Analysis of mutations in the 23S genes that confer antibacterial resistance in Helicobacter pylori.
|Analysis of multiple contiguous CpG sites. Methylation at nine independent CpG sites (highlighted in gray) is quantified in a single Pyrosequencing run. Position-specific information in the context of an analyzed sequence presents broad sequence methylation patterns. Note the built-in quality control sites (highlighted in yellow) consisting of cytosines converted to thymines, demonstrating full bisulfite conversion of the treated DNA.|Detection of tri- and tetra-allelic SNPs can be difficult with commonly used methods. This series of Pyrograms illustrates the ease of Pyrosequencing based detection of a tri-allelic SNP (red outline). C, T and G are serially dispensed in the Pyrosequencing reaction and only the incorporated nucleotides will elicit a signal peak. The result is a different peak pattern for homozygous samples of each allele (upper three Pyrograms) or compound peak patterns for heterozygous samples (lower three Pyrograms).||||||Linearity of methylation quantification by Pyrosequencing. PCR products from varying mixtures of unmethylated genomic DNA and methylated DNA (EpiTect Control DNAs) were analyzed by Pyrosequencing. A tight correlation between the known percentage of methylated DNA in the mixtures (squares) and the methylation percentage reported by Pyrosequencing (triangles) was observed (r2 = 0.9962). The graph represents the quantification of methylation at a single CpG site in the p16 gene.
|The PyroMark Q96 ID manages all steps necessary to rapidly sequence and analyze up to 96 samples. Simply design the necessary assay and run files, load your samples and reagents, and walk away. The PyroMark Q96 ID dispenses reagents and nucleotides to each well with precision and detects emitted light signals with sensitive CCD sensors.||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 96-well plates. New assay design can be done with the PyroMark Q24, which offers the same analysis versatility on a smaller throughput scale.|PyroMark Q96 Software and PyroMark supplementary software are user-friendly interfaces granting access to assay design, run setup, and powerful data analyses of the obtained results. The software are driven by dropdown menus that ensure the correct selection of parameters and analysis modes for any assay, enabling new users to perform Pyrosequencing runs almost immediately.|The PyroMark Q96 Vacuum Workstation enables conversion of PCR products into the single-stranded DNA needed as template for Pyrosequencing. Exposure of the PCR amplicons to a series of optimized solutions denatures and washes the DNA. This process can be carried out for up to 96 samples in parallel and takes only a few minutes.|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.|
Performance

Integrating detection and quantification of genetic variation into one powerful system, Pyrosequencing with the PyroMark platform outperforms other sequence-based solutions in the analysis of targeted short DNA sequences. For analysis of methylation in epigenetic studies, Pyrosequencing generates highly reproducible quantification of methylation frequencies at individual or consecutive CpG sites (see figure "Analysis of multiple contiguous CpG sites"), and can detect and quantify even small changes in methylation levels (see figure "Linearity of methylation quantification").

For genetic testing applications, alleles of variable loci are accurately quantified, and heterozygosity is easily resolved (see figure "Analysis of a tri-allelic SNP"). For microbial identification and resistance typing, Pyrosequencing enables the concurrent analysis of multiple samples for common drug resistance mutations (see figure "Analysis of antibacterial resistance in Helicobacter pylori").

Principle

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.  

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 Q96 ID seamlessly integrates into epigenetics and genetic analysis workflows, and complements QIAGEN's advanced technologies for sample preparation, bisulfite conversion, and PCR amplification. This 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.

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.

Procedure
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.

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 cartridge, 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. The PyroMark Q96 ID 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. In addition, because Pyrosequencing integrates sequence detection and quantification, the high analysis resolution can lead to new discoveries.

Software
Easy-to-use PyroMark Q96 ID Software 2.5 

PyroMark Q96 ID software version 2.5 enables comprehensive analysis of your results. The recently updated software now contains four analysis modes: AQ (allele quantification), SNP (analysis of SNPs and InDels), CpG (methylation analysis), and SQA (sequence identification). These four different types of analyses can be performed on the same plate, in the same run.

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.

特点
参数
海拔 Up to 2000 m (6500 ft)
应用 Methylation analysis, allele quantification (SNP, InDels), sequence analysis
仪器尺寸 490 x 540 x 620 mm (19.3 x 20.5 x 24.4 in)
配合仪器使用的试剂 PyroMark Q96 Tests
运行温度 18–28°C (64–82°F)
过电压保护类别 II
操作地点 For indoor use only
污染水平 2
功率 100–120 V AC, 220–240 V AC; 56–60 Hz
处理温度 28°C (82.4°F) ± 1%
处理时间 10 to 100 minutes for up to 96 samples in parallel
每次处理样本量;通量 1–96
软件 PyroMark Q96 Software (included), PyroMark CpG SW 1.0 (supplementary), PyroMark IndentiFire SW 1.0 (supplementary), PyroMark Assay Design SW 2.0 (supplementary)
技术 Pyrosequencing
重量 52 kg (114.6 lb)

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试剂盒操作手册
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For performing Pyrosequencing reactions on the PyroMark Q96 ID, PyroMark Q96 MD, and PyroMark Q96 MD Automated
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操作软件
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PyroMark Q96 ID Software 2.5 version 2.5.10.7 is the application software for setting up, performing and analyzing runs on PyroMark Q96 ID instruments. The main feature of this release is 64bit compatibility with Windows 7 operating systems.

This software can only be downloaded by users with a registered PyroMark Q96 ID instrument.
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参考文献
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图片
Analysis of antibacterial resistance in Helicobacter pylori
幽门螺杆菌的抗菌性分析。
幽门螺杆菌23S基因的突变会导致其具有抗菌性。
Analysis of multiple contiguous CpG sites
多个连续CpG位点的分析。
多个连续CpG位点的分析。通过一次焦磷酸测序实验,定量分析9个独立的CpG位点(以灰色突出显示)的甲基化状态。被分析序列含有多个甲基化特征。请注意:胞嘧啶残基组成的质控位点(以黄色突出显示)被转换为胸腺嘧啶,显示被处理的DNA已被完全亚硫酸氢盐转化。
Analysis of a tri-allelic SNP
分析三个等位基因的SNP。
使用常规方法分析三个或四个等位基因的SNP(单核苷酸多态性)可能比较困难。这些Pyrogram图表现了使用焦磷酸测序方法检测三个等位基因SNP(红色标出)的便利性。在焦磷酸测序反应中,按顺序加入C、T和G,只有整合的核苷酸会释放光信号得到信号峰值。每个等位基因是纯合子或杂合子,其反应结果具有不同的峰值特点。
Principle of Pyrosequencing – Step 1
焦磷酸测序原理 — 第1步。
Principle of Pyrosequencing – Step 2
焦磷酸测序原理 — 第2步。
Principle of Pyrosequencing – Step 3
焦磷酸测序原理 — 第3步。
Principle of Pyrosequencing – Step 4
焦磷酸测序原理 — 第4步。
Principle of Pyrosequencing – Step 5
焦磷酸测序原理 — 第5步。
Linearity of methylation quantification
甲基化定量分析符合线性特征。
焦磷酸测序定量分析的甲基化呈线性。采用焦磷酸测序技术分析未甲基化DNA和甲基化DNA(EpiTect Control DNAs)的多种混合物的PCR产物。混合物中已知的甲基化DNA百分比(方块)和焦磷酸测序技术测得的甲基化百分比(三角)紧密相关(r2 = 0.9962)。图为对p16基因的一个CpG位点进行甲基化定量检测的结果。
完全整合的系统。
PyroMark Q96 ID可进行快速测序和分析多达96个样本所需的各个步骤。只需设计模板,选择运行模式,将您的样本和试剂上样,仪器可自动完成其余工作。Pyromark Q96 ID会准确的自动分配试剂和核苷酸至每孔中,并通过灵敏的CCD传感器检测光信号。
PyroMark Q96 ID.
符合您需求的仪器。
PyroMark Q96 ID具有灵活的分析模式和高通量特点,每次运行可处理96个样本。而PyroMark Q96 MD可自动处理并追踪多达10个96孔板。PyroMark Q24具有同样的分析模式,但通量略低,可用于设计模板。
软件直观。
PyroMark Q96 Software和PyroMark补充软件具有友好的界面,可用于模板设计、反应体系构建,并为反应所获结果提供强大的分析工具。该软件通过下拉菜单操作,确保正确选择反应参数和分析模式,方便新用户操作焦磷酸测序实验。
高效的模板制备。
PyroMark Q96 Vacuum Workstation可将PCR产物转化为单链DNA用于焦磷酸测序。将PCR扩增子加入多重优化的溶液中进行变性,然后洗涤获得DNA。对96个样本平行进行这一流程,仅需花费几分钟。
工作流程解决方案。
PyroMark Q96 ID可简单、高效的整合入焦磷酸测序工作流程。每一步,从引物设计到PCR扩增和测序模板制备,都由专为焦磷酸测序设计的软件、试剂盒、试剂和样本制备仪器提供高品质支持。