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PyroMark Q48 Autoprep

通过整合模板制备功能进行自动化焦磷酸测序,提升甲基化、突变和SNP定量分析能力
  • 全自动实验流程,减少手工操作
  • 直观的设备和分析软件,简单易用
  • 通量更高,提高单次运行的样本检测数量和每天运行轮次
  • 一流的焦磷酸测序性能,提高测序读长和结果可靠性
  • 使用多引物分配系统,可实现更高的自动化通量
  • 可通过定量DNA甲基化分析,获得更多的研究结果
PyroMark Q48 Autoprep的一大亮点是在焦磷酸测序操作流程中整合了全自动模板制备功能。凭借崭新的设计和软件,该产品将通量在PyroMark Q24 Advanced的基础上提升一倍,同时性能更优异,非常适合各类功能性研究以及NGS和芯片检测实验中的大批量样本验证。
Cat No./ID: 9002470
PyroMark Q48 Autoprep System
PyroMark Q48 Instrument, multistep pipet, software, documentation and installation
Cat No./ID: 9002471
PyroMark Q48 Autoprep Instrument
PyroMark Q48 Instrument, multistep pipet, software and documentation
Cat No./ID: 9002472
PyroMark Q48 Autoprep Priority
PyroMark Q48 Instrument, multistep pipet, software, documentation, installation and 2-year warranty
Cat No./ID: 9002473
PyroMark Q48 Autoprep PrioPLUS
PyroMark Q48 Instrument, multistep pipet, software, documentation, installation and 3-year warranty
Cat No./ID: 9024321
PyroMark Q48 Cartridge Set
Set of all three replacement cartridges for PyroMark Q48 Autoprep instrument; includes Nucleotide Cartridge, Reagent Cartridge and Primer Cartridge
Cat No./ID: 9024322
PyroMark Q48 Nucleotide Cartridge
Replacement PyroMark Q48 Nucleotide Cartridge for PyroMark Q48 Autoprep instrument
Cat No./ID: 9024323
PyroMark Q48 Reagent Cartridge
Replacement PyroMark Q48 Reagent Cartridge for PyroMark Q48 Autoprep instrument
Cat No./ID: 9024324
PyroMark Q48 Primer Cartridge
Replacement PyroMark Q48 Primer Cartridge for PyroMark Q48 Autoprep instrument
Cat No./ID: 9024325
PyroMark Q48 Software License (1)
One additional license for PyroMark Q48 Software. Only valid together with PyroMark Q48 Autoprep
Cat No./ID: 9024326
PyroMark Q48 Software License (5)
Five additional licenses for PyroMark Q48 Software. Only valid together with PyroMark Q48 Autoprep
Cat No./ID: 974230
PyroMark Q48 AutoPrep Starter Kit
PyroMark Q48 Magnetic Beads (300), PyroMark Q48 Advanced CpG Reagents (4 x 48), PyroMark Control Oligo, PyroMark Q48 Discs (50) and PyroMark Q48 Absorber Strips (100)
Cat No./ID: 974901
PyroMark Q48 Discs (50)
50 discs for automated template preparation and Pyrosequencing reactions using a PyroMark Q48 Autoprep
Cat No./ID: 974912
PyroMark Q48 Absorber Strips (100)
100 absorber strips used for liquid collection during automated template preparation with PyroMark Q48 Autoprep

PyroMark Q48 Autoprep适用于分子生物学应用。该产品不适用于疾病的诊断、预防或治疗。


0
Excluding well-to-well and disc-to-disc cross contamination.
In run A, PCR products and non-template controls (NTC) were loaded into a Q48 disc in alternating order. The Pyrosequencing results show no cross-contamination between disc wells. Run B was performed with NTC samples only and without replacing the absorber strip from the first run. The results exclude any cross-contamination from one run to another. Results shown in relative light units.
1
The principle of multiple primer dispension (MPD).
A sequencing primer mixture is loaded into 1 reservoir of the primer cartridge and dispensed to 4 different wells. Each primer only anneals to its specific target sequence and will be elongated during the Pyrosequencing reaction. Primers in each MPD mix should be designed and checked to avoid formation of primer–dimers or binding to another PCR template.
2
Compatibility among PyroMark platforms for mutation analysis.
The NRAS Pyro assay was used to measure mutation frequencies in defined mixtures of wildtype and mutated NRAS sequences. Nine different mixtures representing frequencies of 0, 5, 10, 25, 50, 75, 90, 95, and 100% were analyzed using three different PyroMark platforms. The mutation frequencies measured were plotted against the expected frequencies. The data reveals that all three platforms gave the same results, showing that previously designed assays can be transferred among various PyroMark platforms.
3
Compatibility among PyroMark platforms for methylation analysis.
ER-alpha methylation was measured in defined mixtures of methylated and unmethylated control DNA, representing methylation degrees of 0, 50 and 100%, using four different PyroMark platforms. The methylation measured for one CpG site was plotted against the expected percentage of methylated DNA. All four platforms gave the same results, showing that previously designed assays can be transferred among various PyroMark platforms.
4
Principle of Pyrosequencing – 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.
5
Principle of Pyrosequencing – 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.
6
Principle of Pyrosequencing – 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.
7
Principle of Pyrosequencing – step 4.
Apyrase, a nucleotide-degrading enzyme, continuously degrades unincorporated nucleotides and ATP. When degradation is complete, another nucleotide is added.
8
Principle of Pyrosequencing – 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.
Performance
先进的技术、软件和化学法提高测序读长和结果可靠性

PyroMark Q48 Autoprep进一步改进了试剂和仪器运行算法,同PyroMark Q96和PyroMark Q24系统相比,大幅提高了检测读长和碱基检出、突变分析和甲基化定量分析精确度。以往系统的检测读长会受本底峰值限制,导致测序反应的光信号会有所下降。升级后的PyroMark “Advanced”化学试剂和仪器算法可以减少本底干扰,进而增加读长和结果的可靠性。根据待分析的序列,一次反应可获得140 bp以上的高精度读长。

样本通量更高,提高单次运行处理的SNP和突变检测数量

Multiple Primer Dispensation (MPD) 技术是一种在检测体系数目超过可用卡夹储液槽数目时提高测序引物自动分配能力的策略。PyroMark Q48 Autoprep Primer Cartridge有3个储液槽,但是如果一块测序板上需要进行的检测体系多于3个,那么可以将不同引物混合起来放入同一个引物卡夹内。当模板制备完成后,引物混合物将被自动分配到每个孔内。因为每个孔内只有一种PCR模板,所以只有与其对应的引物可以和模板结合。所有其它引物将留在溶液中不发生结合。MPD混合物中的每个引物都要单独设计和验证,避免其形成引物二聚体或与其它模板结合。

兼容各种PyroMark检测平台

新的PyroMark Q48 Autoprep仪器和“Advanced”化学法直接兼容以往设计的焦磷酸测序检测体系。数据表明,使用不同的PyroMark平台处理同一检测体系时,所获得的突变频率和甲基化分析结果相同。这种跨平台兼容性也不受测序引物中的分析位点距离的影响。这在通过单次反应分析多种序列变异时尤为重要,比如典型的复杂突变体系检测或连续CpG位点甲基化分析。

​新的测序disc孔盘设计避免了磁性模板制备交叉污染

PyroMark Q48 Disc无需用户手动干预即可在同一仪器上完成自动化模板制备和焦磷酸测序。模板制备所需的缓冲液在反应过程中即从样本和disc孔盘中有效地去除,因而每次运行、每个轮次都没有孔间、disc孔盘间的交叉污染风险。

Principle
PyroMark Q48 Autoprep使用基于实时测序的焦磷酸测序技术,在遗传学和表观遗传学研究中进行检测和定量。系统可以同时分析多达48个样本,简单易用的自动化流程制备单链DNA测序模板,无需人工操作。这一流程使用磁性链霉亲和素包被的Sepharose微珠(PyroMark Q48 Magnetic Beads)来结合生物素标记的PCR产物链。系统可以同时自动进行四对测序引物的退火。如果需要用到更多的测序引物,则可以人工将这些引物加入到单链DNA样本中。

​焦磷酸测序反应步骤:

第一步:一个DNA片段被扩增,同时作为焦磷酸测序模板的DNA链被生物素标记。经过变性之后,生物素标记的单链PCR扩增产物被分离出来并于一段测序引物杂交。杂交后的引物和单链模板与DNA聚合酶、ATP硫化酶、荧光素酶、腺苷三磷酸双磷酸酶,以及作为底物的腺苷-5’-磷酸硫酸酐(APS)和荧光素进行孵育。

第二步:将第一个脱氧核苷酸三磷酸(dNTP)加入到反应体系中。如果它和模板链的碱基配对,则在DNA聚合酶催化作用下,该dNTP被加到测序引物末端。每次合成事件均伴随一个焦磷酸(PPi)的释放,释放的焦磷酸摩尔数等于合成的碱基数。

第三步:在存在APS的条件下,ATP硫化酶将PPi转化为ATP。生成的ATP驱动萤光素酶将荧光素转化为氧化荧光素,同时产生与ATP数量正相关的可见光。在荧光素酶催化反应中产生的可见光被PMT探测器检测到并记录原始数据中的一个峰(Pyrogram)。每个峰(即光信号)的高度与合成的碱基数正相关。

第四步:腺苷三磷酸双磷酸酶(一种核苷酸降解酶)持续降解未结合的核苷酸和ATP。当降解完成后,另一个核苷酸被加入到反应体系中。

第五步:顺序依次加入dNTP。需要注意的是在反应体系中使用脱氧腺苷α-硫代磷酸(dATPαS)代替了天然的脱氧腺苷磷酸(dATP),因为dATP虽然能被DNA聚合酶有效使用,但不能被荧光素酶识别。随着这一过程的进行,互补DNA链逐渐延长,同时Pyrogram轨迹记录的信号峰值反映出核酸的序列。
Procedure
PyroMark Q48 Advanced软件生成一个运行文件并通过USB接口或网络连接传输到仪器中。试剂、核苷酸和缓冲液按照触摸屏上显示的体积被加入到PyroMark Q48 Autoprep测序槽中。生物素标记的PCR产物和磁性链霉亲和素包被的Sepharose微珠被加入到Q48 Disc孔盘的孔内,然后将孔盘和吸收条放入仪器内。单链模板的制备和测序引物的退火现在已经完全自动化,不需要额外的人工操作。仪器可自动分配3种不同测序引物或Multiple Primer Dispensation (MPD)混合物。可选择性的人工加入额外的测序引物,进一步提升了单次运行可检测的样本数。
Applications
焦磷酸测序在多个学科的研究中的应用价值日益突出。无论是检测病原体的抗药性演化,DNA甲基化对基因表达的表观遗传调控,家畜中特定表型的遗传标记,还是法医样本中的线粒体DNA多态性,PyroMark平台都能提供强大而灵活的遗传和表观遗传变异分析。此外,焦磷酸测序整合了序列检测和定量功能,其强大的分析能力更易促成新的发现。
Software
PyroMark Q48 Autoprep Software安装于PC上,可对您的结果进行全面的分析。该软件包括4种分析模式——AQ, SNP, CpG和SEQ。AQ模式可用于对多种突变SNP和InDel进行定量研究。它适用于分析单个或多个突变位点,以及双拷贝、三拷贝或四拷贝等位基因突变。SNP模式提供对SNP和InDel的基因型分析。CpG模式可以分析单个或多个CpG或CpN位点,并提供针对亚硫酸氢盐处理的内参。SEQ用于对未知序列的碱基序列进行解读。

​PyroMark Q48 Autoprep Software是一款用户友好、直观的软件,提供方便易用的先进分析方法。如果运行中出现问题,或系统检测到实验的不一致性,软件或对每个孔提出针对性的警告信息。点击"Warning Info"按钮可以获得有关警告的进一步信息和排除故障的建议以及如何避免在后续实验中再次出现该问题。
Features
Specifications
应用 Simultaneous running of multiple assays and assay types including SNP, AQ, CpG and SQA to achieve methylation analysis, de novo sequencing, mutation characterization including In/Dels, speciation, quantitative allele sequencing and SNP genotyping
连接 One USB port, Ethernet connection
湿度 Relative humidity of 20–80% (noncondensing)
仪器尺寸 W X D X H: 250 mm (9.8 in.) X 300 mm (11.8 in.) X 300 mm (11.8 in.) with chamber lid and injector cover closed; 250 mm (9.8 in.) X 560 mm (22 in.) X 390 mm (15.4 in.) with chamber lid and injector cover open
配合仪器使用的试剂 PyroMark Q48 Advanced Reagents, PyroMark Q48 Advanced CpG Reagents
运行温度 18–30ºC (64–86ºF)
过电压保护类别 II
操作地点 For indoor use only in a draft-free location. No exposure to direct sunlight.
污染水平 2
功率 100–240V AC, 50/60 Hz. Mains supply voltage fluctuations are not to exceed ±10% of the nominal supply voltages.
处理温度 28°C ± 0.5°C
处理时间 Depends on the number of dispensations
每次处理样本量;通量 1–48
软件 PyroMark Q48 Advanced Software (to be installed on PC)
技术 Pyrosequencing
重量 8.5 kg (18.7 lb)
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