适用于在长序列测序中利用焦磷酸测序实现先进的甲基化和突变定量
  • 先进的技术、软件和化学试剂,可用于长序列分析
  • 连续CpG或CpN位点的定量甲基化分析
  • 任意序列位置的改进的序列差异定量
  • 简单且更佳的碱基检出功能
  • 同一仪器和同一运行中的Assay通用性
PyroMark Q24 Advanced改进了焦磷酸测序技术,可提供比以往更佳的实时测序检测和定量。PyroMark Q24 Advanced采用先进的技术、软件和化学试剂,适用于分析各种类型的序列差异,尤其是CpG或CpN位点的DNA甲基化、复杂突变或用于从头测序应用(如微生物分型)。
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PyroMark Q24 Advanced System
Instrument and software for advanced Pyrosequencing analysis: includes installation, training, and 1-year warranty on parts and labor
9002270
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PyroMark Q24 Advanced Priority Package
Instrument and software for advanced Pyrosequencing analysis: includes Priority Package with installation, training, 2-year warranty on parts and labor, and 2 preventive maintenance visits
9002272
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PyroMark Q24 Advanced Priority Package Plus
Instrument and software for advanced Pyrosequencing analysis: includes Priority Package Plus with installation, training, 3-year warranty on parts and labor, and 3 preventive maintenance visits
9002273
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PyroMark Q24 Advanced Software
Analysis software for upgrading PyroMark Q24 systems
9022779
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PyroMark Q24 Advanced和PyroMark Q24 Advanced 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 Advanced、PyroMark Q24 Advanced 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。
Analysis of 16 CpG sites in a long sequence run.|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.|Improved methylation quantification in homopolymers.|Long de novo sequencing runs.|Quantitative mutation analysis in long sequence runs.|Workflow solutions.|Fully integrated system.|Easy data management.|Efficient template prep.|
PyroMark Q24 Advanced increases both read length and reliability of methylation analysis at positions later in the sequence. This example demonstrates 135 nucleotide dispensations and the accurate analysis of 16 different CpG positions in a single PyroMark Q24 Advanced CpG reaction. To accurately analyze all of these sites with PyroMark Q24, one would need to run 3 separate assays.|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.|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.|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.|Apyrase, a nucleotide-degrading enzyme, continuously degrades unincorporated nucleotides and ATP. When degradation is complete, another nucleotide is added.|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.|Quantification of CpG methylation directly following a T homopolymer or between T homopolymers is especially challenging. Methylation levels are determined by the ratio of converted C nucleotides to unconverted C nucleotides following bisulfite treatment, and since the C to T conversion often leads to long T homopolymer stretches in an amplicon, this scenario occurs often. PyroMark Q24 Advanced enables accurate quantification of CpG position after or between T homopolymers.This example shows the analysis of a CpG site within a stretch of 8 T nucleotides.|Read length is a critical factor for analysis of unknown sequences, often limiting the reliable analysis to 40–80 bases. The improved chemistry and algorithm of PyroMark Q24 Advanced significantly increases the possible read length and provides higher accuracy. PyroMark Q24 (upper panel) and PyroMark Q24 Advanced (lower panel) were used for de novo sequencing using the same assay. Blue bars indicate reliably detected bases; yellow indicates bases that might have been determined correctly but should be checked by the user; red indicates unreliable readings.|Since mutations and single nucleotide polymorphisms (SNPs) are rarely in directly neighboring positions, common Pyrosequencing chemistry might require separate assays for analysis of more than one mutation site. The new chemistry of PyroMark Q24 Advanced allows much longer runs, enabling reliable analysis of additional mutations within one run. This example shows the analysis of a 10:90 mixture of wild-type and mutated EGFR. Even after 60 dispensations, the analysis is exact.|The components of the PyroMark Q24 Advanced 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, PyroMark Q24 Advanced 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 instrument 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.|PyroMark Q24 Advanced 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 Advanced 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.|
Performance
较长的焦磷酸测序运行和更高的准确度
PyroMark Q24 Advanced采用了改进的化学试剂和仪器操作算法,大大增加了读取长度和碱基检出功能的准确度,可轻松实现较长的从头测序运行分析。此前,读取长度受到测序反应中的背景峰和较低的光信号的限制。已升级的PyroMark Q24 Advanced化学试剂和算法可降低该背景峰,从而提高读取长度和可靠性。根据待分析序列的不同,只需一次PyroMark Q24 Advanced反应即可获得高度准确的140个或更多碱基的读取长度 (参见Long de novo sequencing runs)。
任意位置的更先进的甲基化分析
PyroMark Q24 Advanced可在任意序列位置实现改进的长片段序列甲基化定量。以前,远离测序引物的甲基化位点分析是无法确定的,但现在利用更长的读取长度和更高的准确度,可在整个测序运行中实现高度可靠的甲基化定量(参见Analysis of 16 CpG sites in a long sequence run)。
改进的同聚物测序准确度
DNA甲基化分析中的亚硫酸氢盐转化可导致核苷酸序列中频繁的poly T延伸,以前,由于这些位点的光信号定量的不确定性,在这种T同聚物之后直接进行CpG位点分析面临诸多难题。PyroMark Q24 Advanced具有更高的准确度,可在8个T核苷酸链之后甚至之中实现可靠的CpG甲基化定量(参见Improved methylation quantification in homopolymers)。
长片段序列的突变分析
PyroMark Q24 Advanced还可利用单个Assay实现可靠的多态性定量。由于单核苷酸多态性(SNP)及其他突变通常不相邻,因此传统的焦磷酸测序化学方法一般需要分析针对每个突变位点的单独的Assay。全新的PyroMark Q24 Advanced化学方法可实现更长片段分析,在一次运行中实现多个突变或SNP的可靠分析(参见Quantitative mutation analysis in long sequence runs)。
Principle
焦磷酸测序技术采用了合成测序原理,可在数分钟内提供序列定量数据。PyroMark Q24 Advanced是一套全集成系统,可提供实时序列信息,具有高准确度和长读取长度,尤其适用于复杂突变、表观遗传学研究、抗性分型和微生物鉴定分析。该系统包括PyroMark Q24仪器(运行固件3.0或更高)、PyroMark Q24 Advanced Software、PyroMark Q24 Advanced Reagents/PyroMark Q24 Advanced CpG Reagents、PyroMark Q24 Vacuum Workstation和PyroMark Control Oligo(参见下表)。此外还提供样本制备解决方案,利用PyroMark Q24 Vacuum Workstation实现单链DNA的制备。 
产品 描述
PyroMark Q24 适用于定量序列分析测序仪器
PyroMark Q24 Advanced Software 包括4种分析模式的分析软件(AQ:等位基因定量;SNP:基因分型;CpG:CpG或CpN位点的甲基化;SEQ:未知序列的碱基检出)
PyroMark Q24 Advanced Reagents 酶、底物、核苷酸和缓冲液。仅适用于利用PyroMark Q24 Advanced Software设置和分析的Assay。
PyroMark Q24 Advanced CpG Reagents 酶、底物、核苷酸和缓冲液,适用于要求较长序列读取长度的Assay。仅适用于利用PyroMark Q24 Advanced Software设置和分析的Assay。
PyroMark Q24 Vacuum Workstation 可同时制备至多24个样本的工作站(从PCR产物到单链模板)
PyroMark Q24 Control Oligo 确认安装和系统操作正确的对照品

焦磷酸测序反应步骤:
步骤1:扩增DNA区段,对用作焦磷酸测序模板的DNA链进行生物素化。变性后,分离生物素化的单链PCR扩增片段,并将其与测序引物杂交。将杂交引物和单链模板与DNA聚合酶、ATP硫酸化酶、荧光素酶和三磷酸腺苷双磷酸酶及底物腺苷-5'-磷酰硫酸(APS)和荧光素一起孵育(参见Principle of Pyrosequencing — step 1)。

步骤2:将第一个三磷酸脱氧核苷(dNTP)加入反应中。DNA聚合酶催化dNTP加至测序引物上(若其与模板链上的碱基互补)。每次结合均伴有焦磷酸(PPi)释放,并与加入的核苷酸呈等摩尔量(参见Principle of Pyrosequencing — step 2)。

步骤3:在腺苷-5'-磷酰硫酸(APS)存在的条件下,ATP硫酸化酶可将PPi转化为ATP。ATP促进了荧光素酶介导的荧光素至氧化荧光素的转化,产生的可见光与ATP的量成正比。利用CCD传感器检测荧光素酶催化反应生成的光,在原始数据中呈峰状(Pyrogram)。各个峰的高度(光信号)与加入的核苷酸数成正比(参见Principle of Pyrosequencing — step 3)。

步骤4:三磷酸腺苷双磷酸酶——一种核苷酸降解酶——可不断降解未结合的核苷酸和ATP。当降解完成后,加入其他核苷酸(参见Principle of Pyrosequencing — step 4)。

步骤5:顺序加入dNTP。应注意的是,deoxyadenosine alpha-thio triphosphate(dATPαS)可为DNA聚合酶有效利用,而荧光素酶无法识别,因此被用作天然脱氧腺苷三磷酸(dATP)的替代物。随着过程的继续,互补DNA链不断延伸,利用Pyrogram示踪图上的信号峰确定核苷酸序列(参见Principle of Pyrosequencing — step 5)。
简化工作流程——从样本到结果
通用PyroMark Q24 Advanced无缝整合至表观遗传学和基因分析工作流程,可与QIAGEN先进的样本制备、重亚硫酸盐转化和PCR扩增技术形成互补。高度可靠的仪器可实现CpG或CpN位点及突变的序列检测和甲基化定量。采用简化的工作流程,可以更快速地获得结果。
Procedure
快速方便的样本制备
从PCR产物到适合测序使用的单链模板——采用PyroMark Q24 Vacuum Workstation可同时制备至多24个样本,仅需不到15分钟。工作站操作简单,实际“操作时间”不到5分钟。

在焦磷酸测序之前,先生成生物素化PCR产物。该生物素化PCR产物可与链霉亲和素包被的Sepharose微珠结合,利用Vacuum Workstation上的Vacuum Tool可捕获微珠,彻底冲洗并变性,生成适用于焦磷酸测序的单链DNA。将上述模板DNA加至包含测序引物的焦磷酸反应板中,引物退火后,将反应板置于PyroMark仪器上。PyroMark Q24 Advanced Reagents或PyroMark Q24 Advanced CpG Reagents包含焦磷酸测序反应所需的酶、核苷酸和底物;参照软件提供的体积,将试剂加至分装装置内,同样置于仪器内供焦磷酸测序使用。
Applications
焦磷酸测序在各学科的研究应用中发挥了愈来愈重要的作用。不论是检查病原体中的抗药性的产生、基因表达调控中表观遗传学DNA甲基化的作用、家畜中特定表型的遗传标志物,还是线粒体DNA法医样本中的多态性,PyroMark Q24 Advanced均可提供功能强大且通用的基因和表观遗传学变异分析。此外,由于焦磷酸测序整合了序列检测和定量,提高的分析分辨率可促进新发现的诞生。

产品对比
PyroMark Q24 Advanced PyroMark Q24
通量 1–24 个样本 1–24个样本
体积 25 µl 25 µl
PCR产物需要量 5–10 µl
(~0.5–3 pmol产物)
5–10 µl
(~0.5–3 pmol产物)
读长(取决于Assay和序列) 10–140 bp以上 10–80 bp
应用软件 PyroMark Q24 Advanced SW
(需要固件1.5.6903或更高)
PyroMark Q24 SW 2.0
软件功能 SEQ(从头测序)
CpG/CpN甲基化
SNP
AQ
SQA (从头测序)
CpG甲基化
AQ/SNP
主要应用 复杂突变分析
表观遗传学(CpG和CpN分析)
耐药性分析和微生物鉴定
突变分析
耐药性分析
兼容试剂 PyroMark Q24 Advanced Reagents
PyroMark Q24 Advanced CpG Reagents
PyroMark Q24 Gold Reagents
灵敏度 2%的突变
98% wt
2%的突变
98% wt
Software
 易于使用的PyroMark Q24 Advanced Software
在PC上安装PyroMark Q24 Advanced Software,可对您的结果进行全面分析。该软件包括4种分析模式——AQ、SNP、CpG和SEQ。AQ模式适用于各种突变定量研究,如SNP和InDels。它适用于单变量和多变量位点及双、三和四等位基因突变分析。SNP模式提供了SNP和InDels的基因型分析。CpG模式可实现单一或多个CpG或CpN位点的甲基化分析,并提供了重亚硫酸盐处理的内部对照。SEQ模式适用于未知序列的碱基检出。

PyroMark Q24 Advanced Software使用方便且直观,为运行分析提供了改进的便捷工具。如果在运行过程中出现问题,或者如果系统利用Assay检测出不一致性,则软件可针对各单独孔提供特定的警告信息(参见警告信息和建议)。“警告信息”(Warning Info)按钮可提供其他警告信息及疑难解答建议和防止其在后续Assay中发生的建议。
使用PyroMark Assay Design Software,灵活、简单的设计焦磷酸测序实验
采用PyroMark Assay Design Software 2.0可轻松设计PCR和测序引物进行焦磷酸测序分析。Assay已经过优化,适用于所有PyroMark仪器。
Feature
Specifications
Altitude Up to 2000 m (6500 ft)
Applications Methylation analysis (CpG and CpN sites), allele quantification, genotyping, sequence analysis, microbial typing
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 Advanced Reagents, PyroMark Q24 Advanced CpG Reagents
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
Samples per run; throughput 1–24
Software PyroMark Q24 Advanced Software (to be installed on PC)
Technology Pyrosequencing
Weight 27.5 kg (60.6 lb)

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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0003
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0004
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0005
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0006
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0007
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0008
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0009
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0010
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0011
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0012
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0013
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0015
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Instrument methods file for use with PyroMark Q24 Advanced Software, PyroMark Q24 Advanced Reagents or PyroMark Q24 Advanced CpG Reagents, and PyroMark Q24 Cartridges marked with method 0014
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Files for upgrading existing PyroMark Q24 instruments to firmware 1.5.6903
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Operating Software
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PyroMark Q24 Advanced Software version 3.0.1 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 Advanced Software license and registered PyroMark Q24 Advanced instrument. If you do not have a valid software license, contact your QIAGEN sales representative.
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Files for upgrading existing PyroMark Q24 instruments to firmware 1.5.6903
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