应用焦磷酸测序技术定量分析遗传和表观遗传的DNA变化

  • 在同一台仪器上对同一批样品进行多功能分析
  • 可靠的定量分析等位基因和甲基化状态
  • 序列信息帮助发现稀有突变
  • 15分钟内平行分析1–24个样本
  • 紧凑的检测平台,占用空间最小

PyroMark Q24应用焦磷酸测序技术,对序列突变和表观遗传学甲基化进行实时的基于序列的检测和定量。PyroMark Q24非常适合用于分析CpG位点甲基化、SNPs、插入/缺失、STR、基因拷贝数变异以及微生物鉴定和抗性分型。

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PyroMark Q24 System
Instrument and software for Pyrosequencing analysis: includes installation, training, and 1-year warranty on parts and labor
9001514
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PyroMark Q24 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
9001913
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PyroMark Q24 Priority Package Plus
Instrument and software for Pyrosequencing analysis: includes Priority Package Plus with installation, training, 3-year warranty on parts and labor, and 3 preventive maintenance visits
9001914
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PyroMark Q24 Software 2.0
Analysis software, for laboratory use only
9019062
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PyroMark Q24 Instrument和PyroMark Q24 Software应与相应的QIAGEN试剂盒配合使用,适用于试剂盒用户手册中指定的应用。如果将PyroMark Q24 Instrument、PyroMark Q24 Software与非QIAGEN的试剂盒配合使用,用户应自行确认是否适用于相关应用。


CpG methylation analysis of the MLH1 gene.|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.|CpG methylation pattern in the RASSF1A gene.|Workflow solutions.|Fully integrated system.|Easy data management.|Efficient template prep.|The right instrument for your needs.|
Highlighted areas in the Pyrogram trace indicate variable CpG positions (light gray) and built-in bisulfite treatment controls (yellow). The methylation level of each CpG site is indicated in blue boxes on top of the Pyrogram trace. Data kindly provided by Dr. Triantafillos Liloglou, Roy Castle Lung Cancer Foundation, Molecular Oncology, Liverpool.||||Apyrase, a nucleotide-degrading enzyme, continuously degrades unincorporated nucleotides and ATP. When degradation is complete, another nucleotide is added.||The figure illustrates the variation in methylation level between 5 different CpG sites in 4 individual tumor samples. Each sample was run in duplicate, and the concordance between the two samples clearly illustrates the reproducibility of Pyrosequencing technology.|The components of the PyroMark Q24 System are designed to make the Pyrosequencing workflow straightforward and efficient. Each step, from assay design to PCR amplification and preparation of sequencing templates, is supported by software, kits, reagents, and sample prep instrumentation optimized for Pyrosequencing.|Though small in size, the PyroMark Q24 manages all steps necessary to rapidly analyze up to 24 samples. Simply load your samples and reagents, upload your run file, and walk away. The PyroMark Q24 dispenses reagents and nucleotides to each well with precision. Light signals emitted are detected by 24 CCD sensors — one sensor per well — thereby eliminating signal crossover.|The PyroMark Q24 is designed as a stand-alone instrument, which makes it easy to place anywhere in the lab. Data are stored on the instrument hard drive and can be viewed on the instrument screen during a run. Additionally, all files are stored on the supplied USB stick, giving the user the flexibility to analyze data on any computer with PyroMark Q24 Software installed.|The PyroMark Q24 Vacuum Workstation enables conversion of PCR products into the single-stranded DNA for Pyrosequencing. Exposure of the PCR amplicons to a series of optimized solutions denatures and washes the DNA. This process is carried out for 24 samples in parallel and takes only a few minutes.|While the PyroMark Q24 combines ease-of-use, analysis versatility, and superior detection sensitivity, your workflow may demand higher throughput. The PyroMark Q96 ID and PyroMark Q96 MD accommodate this need by processing up to 96 samples in a single run. Furthermore, the automation options of the PyroMark Q96 MD enable hands-free processing of up to ten 96-well plates.|
Performance
高度适用于表观遗传学研究的检测工具

焦磷酸测序技术配合QIAGEN表观遗传学产品线,提供高度可靠的序列数据,能够准确、灵敏的定量分析甲基化水平(参见"CpG methylation analysis of the MLH1 gene")。甚至能够检测新的突变,以及低水平的DNA异常甲基化。PyroMark Q24包括了一个完整软件包,用于分析CpG位点甲基化,以及用于确认亚硫酸氢盐处理彻底与否的内置的对照。

定量分析单个CpG位点

分析单个CpG位点对于研究各种肿瘤基因的异常表达是非常重要的(参见"CpG methylation pattern in the RASSF1A gene")。由于传统方法获得的数据分辨率低,通常灵敏度不高。焦磷酸测序技术克服了这一局限,可高度精确地分析有一个或多个CpG位点的甲基化序列单个位点的变化。

Principle

焦磷酸测序技术基于测序原理,数分钟内可获得定量数据。PyroMark Q24是提供实时测序信息的完整体系,高度适用于表观遗传学研究和遗传分析。该系统包括PyroMark Q24、PyroMark Q24 Vacuum Workstation、PyroMark Q24 Software、PyroMark Gold Q24 Reagents、PyroMark Control Oligo和PyroMark Q24 Validation Oligo(见表)。同时提供样本制备方案,可使用PyroMark 24 Vacuum Workstation制备单链DNA。

 体系组成 描述
PyroMark Q24 用于定量分析突变和甲基化的测序分析仪器
PyroMark Q24 Vacuum Workstation 可平行制备24个样本的工作站
PyroMark Q24 Software 分析软件,提供2种分析模式(用于CpG位点分析和等位基因定量)
PyroMark Q24 Gold Q24 Reagents 酶、底物和核苷酸
PyroMark Q24 Control Oligo 对照,用于确定体系的正确安装和运行
PyroMark Q24 Validation Oligo 对照,用于确定体系性能
PyroMark Q24体系
高度适用于遗传分析的平台

遗传分析涵盖多种分析基因组DNA差异的应用,包括突变检测和SNP分型。PyroMark Q24可精确、高度灵敏地分析靶基因突变和定量分析混合细胞群体的等位基因频率。QIAGEN采用焦磷酸测序技术为基因突变分析提供经优化和验证的RUO检测。

焦磷酸测序反应步骤:

步骤1:扩增DNA片段,作为焦磷酸测序模板的单链是生物素标记的。变性后,分离生物素标记的单链PCR扩增子,与测序引物杂交。杂交的引物和单链模板同各种酶,包括DNA聚合酶、ATP硫酸化酶、荧光素酶、三磷酸腺苷双磷酸酶以及底物腺苷酰硫酸(APS)和荧光素一起孵育(参见"Principle of Pyrosequencing — step 1")。

步骤2:第一个脱氧核苷三磷酸(dNTP)加入反应中,在DNA聚合酶的作用下,dNTP结合到DNA链上与其互补的碱基位。每次结合都会释放与核苷酸相等摩尔量的焦磷酸(PPi)(参见"Principle of Pyrosequencing — step 2")。

步骤3:在有APS的条件下,ATP硫酸化酶将PPi转化为ATP。在这个ATP的驱动下,荧光素酶将荧光素转化为氧化荧光素,并产生可见光,可见光的量与ATP的量成正比。通过电荷耦合(CCD)相机即可检测到荧光素酶催化反应产生的可见光,并在原始数据输出(Pyrogram)中显示为一个峰,每个峰的高度(光信号)与结合的核苷酸数量成正比(参见"Principle of Pyrosequencing — step 3")。

步骤4:三磷酸腺苷双磷酸酶,是一种核苷酸降解酶,会降解未结合的核苷酸与ATP。完全降解后,加入另一个核苷酸(参见"Principle of Pyrosequencing — step 4")。

步骤5:持续添加dNTP。α-硫代脱氧腺苷三磷酸(dATPαS)在反应中用于替代天然脱氧腺苷三磷酸(dATP),这是因为DNA聚合酶可高效的应用前者,而不被荧光素酶识别。当该过程连续进行时,就会形成互补的DNA链,可根据Pyrogram追踪的信号峰确定核苷酸序列(参见"Principle of Pyrosequencing — step 5")。

简化的工作流程—从样本到结果

多功能PyroMark Q24与表观遗传学和遗传学分析流程无缝接合,且与QIAGEN先进的样本制备、亚硫酸氢盐转化和PCR扩增技术兼容。借助于高度可靠的仪器可在测序水平上对CpG位点和突变进行检测和定量分析。简化的工作流程可更快速地获得结果。

Procedure
快速方便的样本制备

从PCR产物到直接用于测序的单链模板,应用PyroMark Q24 Vacuum Workstation可在15分钟内同时制备24个样本。该工作站操作简单,且手动时间少于5分钟。

在焦磷酸测序之前,形成了一个生物素标记的PCR产物,该产物结合到覆盖链酶亲和素的琼脂糖凝胶珠上。这些胶珠会被真空仪器捕捉并彻底清洗,随后变性,得到适合于焦磷酸测序的单链DNA。该模板DNA放入含测序引物的焦磷酸测序反应板中,随后引物退火,再将该板放入PyroMark仪器中即可。PyroMark Gold试剂包含用于焦磷酸测序反应的酶、核苷酸和底物,将这些试剂加入分配吸头或卡夹(取决于仪器),按照软件中提供的体积,试剂被加入到仪器中,用于焦磷酸测序。

Applications

焦磷酸测序技术对各个领域的研究应用至关重要。焦磷酸测序技术能够灵敏的分析遗传学和表观遗传学的变化,如检测病原体中药物抗性的发展,基因表达分析中表观遗传学DNA甲基化的作用、牲畜特异性表现型的遗传标记,以及法医检材样本中线粒体DNA的多态性。此外,由于焦磷酸测序技术整合了序列检测和定量,提高了分析灵敏度,助于发现新的信息。

Software
 易于使用的PyroMark Q24软件

安装在电脑上的PyroMark Q24软件能够全面分析您的结果。该软件包含两个分析程序:CpG和AQ(等位基因定量)。两个程序可在同一块板上分析样本,并且能够同时检测不同类型的样本。AQ程序用于分析单个和多个位点,以及双、三和四等位突变。CpG程序能够分析多个连续的CpG位点,并提供亚硫酸氢盐处理的内参。

使用PyroMark Assay Design Software,灵活、简单的设计焦磷酸测序实验

PyroMark Assay Design Software 2.0可简单的设计PCR和测序引物。这些产品是经过优化的,可用于所有的PyroMark仪器。

Feature
Specifications
Altitude Up to 2000 m (6500 ft)
Applications Methylation analysis, allele quantification, genotyping, sequence analysis
Chemical resistance pH 4 to pH 9, common detergents, 0.5 M sodium hydroxide, ethanol
Connections One USB port (2.0)
Humidity Relative humidity of 20–90% (noncondensing)
Instrument dimensions 420 x 390 x 525 mm (16.5 x 15.4 x 20.7 in)
Kits designed for this instrument PyroMark Q24 Tests
Operating temperature 15–32°C (59–90°F)
Overvoltage category II
Place of operation For indoor use only
Pollution level 2
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)
Technology Pyrosequencing
Weight 27.5 kg (60.6 lb)

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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.
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