这项研究是由研发项目为国家研究基金会协会（NRF），通过科学，信息和通信技术及未来规划（批准号:2013M3C8A1075908）部资助支持。

1. DNA提取FFPE参考标准细胞系注意:对于这个过程，DNA提取自FFPE参考标准细胞系（HD598，HD593，HD617，HD273和野生型（WT））使用FFPE组织DNA分离试剂盒，如以下方案中所述进行。自动DNA提取按照制造商的总DNA分离指令来实现的。 <ol><li>使用切片机和原始FFPE块，前手工DNA提取和分析准备新鲜的部分，按照既定的程序。确保，所述组织部分（多个）样本的输入分析的不超过10微米的组合总厚度和表面面积不超过600毫米2为一个节。 </li></ol> 1.2 TPS协议注意:在表1所示的量对应处理48个样品所需要的最低限度，并且过程示出的是按照在TPS准则。在实验开始之前沉淀下来的电子管FFPE样品离心600 XG，以避免自动程序过程中样品的损失。 <ol><li>打开自动DNA分离仪和计算机。打开运行控制软件并插入自动加载托盘到TPS甲板装卸区。 </li><li>分配的试剂成其相应的槽，如图1。 <ol><li>放置4个样品（BRAF WT，BRAF V600E 50％，5％和1％）的样品载体机架。 </li><li>将吸头盒在第2和第3的槽，并检查提示每尖端多个过滤器的存在，这将中止运行。 </li><li>确保裂解缓冲液和洗涤缓冲液的适当的混合通过反转他们3-5倍并将它们加载到在塔4（图1）的各个槽。 </li><li>反转的几十倍或轻度vort后exing，加载洗脱缓冲液，磁珠，并在中柱5的小槽的FFPE缓冲器，留下1个时隙空指示的位置（图1， 表1）。 </li><li>加载2ml的深孔板（DWP）到板载体（ 图1）。 </li></ol></li><li>执行以下最后步骤开始运行之前: <ol><li>摘帽所有试管和试剂的低谷。确认足够的容量可在废液瓶。确认固体废弃物酒瓶是空的，内衬生物危害袋。确认该尖端排出板集中在废组件。 </li><li>关闭前盖。 </li></ol></li><li>启动软件。打开NA_Prep_Main_MLSTARlet.med文件。 </li><li>点击"开始"。仪器状态将从空闲切换到运行。 </li><li>输入样本的数目为此运行。选择适合该运行（DNA = 0）所需的方法。输入第一个大体积的位置UME提示，选择"1"，如果所有的盘都满了。进入第一标准体积尖端的位置，选择"1"，如果所有托盘已满。 注:然后，仪器将通过自动化的步骤运行，而无需用户干预。详细的工作流程示于图2，一旦实验结束时，试剂的浪费和尖端注入废物组件。 </li><li>通过使用荧光法定量纯化的基因组DNA。 注意:含有3.3毫微克/微升的最小浓度的DNA样品进行ddPCR分析（第2部分）。 </li></ol> 2. DNA突变剖析:ddPCR协议注意:该协议用于DNA突变谱由三个主要步骤:1）液滴代，2）常规PCR扩增，3）液滴阅读和4）的DNA的突变谱。 2.1。液滴产生注:ddPCR SuperMix混合是推荐ddPCR，因为这个组合中包含所需的墨滴产生试剂。 <ol><li>为了避免污染，遵循标准预防措施，如戴手套，用干净的PCR罩，洁净移液管，低蛋白结合管。 </li><li>确保人类基因组DNA样本的最小浓度为3.3纳克/微升。注意:纯化的DNA试样的浓度的量将基于％突变频率检测的分析而​​变化。 </li><li>装配在96孔PCR板的反应混合物。解冻并平衡反应组分至RT。通过结合2X ddPCR SuperMix混合（10微升）和20引物（正向和反向，900纳米）和探​​头（250纳米）与每个纯化DNA样品（66ng / 2微升）使达到20微升蒸馏水制备PCR反应（如每个液滴发生器协议） </li><li>彻底涡混合以确保均匀和短暂离心分配之前收集在试管底部的内容。装载20＃181，L到墨盒液滴形成。 </li><li>操作液滴发生器，根据制造商的推荐方案<ol><li>将墨盒插入与定位在保持器的上部左侧的盒凹口的保持器。加入20微升的反应混合物含有样品到中间的，和70微升发生器油进入底部的孔中。 </li><li>附上垫片横跨卡盒的顶部。确保垫圈是在支架的两端牢固地钩住;否则，足够的压力为产生液滴将无法实现。 </li><li>按仪器顶部的绿色按钮，打开液滴生成，然后插入。当支架是在正确的位置，这两个电源（左右）和保持器（中间右）指示灯是绿色的。 </li><li>按下顶部的按钮，仪器再次关闭大门，并开始产生液滴。注:按b后utton，歧管自身定位在出口井，通过微流体通道，其中创建液滴拉伸油和样品。水滴流入滴好，在那里积累。液滴指示灯（右）绿色闪烁10秒后，表示液滴产生正在进行中。 </li><li>当液滴生成完成后，所有的3个指示灯变为绿色常亮;通过按下按钮打开车门，并从单位取出固定器。从支架上取下一次性垫圈并丢弃。注意:暗盒的顶端孔中含有液滴，中，下孔几乎空的，用少量的残余油。 </li></ol></li></ol> 2.2。为PCR准备<ol><li>对于每个样品，吸管出40微升从顶部以及墨盒液滴内容插入到推荐的96孔PCR平板的单个以及在制造商的仪器协议指示。否德:使用多通道移液器是理想的转印液滴乳液。水滴缓慢而温和的愿望，建议尽量减少在传输过程中液滴剪切。 </li><li>转移滴，以避免蒸发后，立即密封PCR板带箔。使用可刺穿箔片密封件是与PCR板密封件并在液滴读者针兼容。按照在PCR板封口机使用说明书中的说明。 </li><li>设置板密封温度180℃，时间为5秒。 </li><li>轻触箭头，打开纸盒门。放置支撑块的96孔的一面朝上放在托盘上。将96孔板在载体块，并确保所有板孔与支撑块对齐。 </li><li>用1片材的可刺穿箔密封覆盖96孔板。一旦96孔板被固定在支撑块上，并覆盖上可刺穿箔密封，触摸密封按钮。托盘将关闭和热封将启动。 </li><li>当热密封完成后，门自动打开;从热循环加热块取出盘子，然后取出加热块。 </li><li>确保所有的井板通过检查，在箔洼地超过每显而易见密封良好。密封后，该板块已准备好热循环。 </li><li>一旦液滴被除去，压在盒保持器的闩锁以将其打开。取出空墨盒并将其丢弃。 </li><li>通过以下在表2中详述的参数执行常规PCR扩增。 </li></ol> 2.3微滴读数（根据制造商建议的协议） 注意:继核酸靶的液滴中PCR扩增，液滴读者仪器分析单独使用2色检测系统 13的每个微滴。我们通常设置为检测FAM的ðVIC记者荧光。 <ol><li>点击"刷新系统"素滴阅读器，并使其准备ddPCR分析。 </li><li>板装入液滴阅读器，然后单击"开始"。液滴读者吸取每一个样品，singulates的液滴和液流他们鱼贯过去双色探测器。检测器读出的液滴来枚举阳性和阴性样品。 </li><li>当液滴阅读完毕后，打开车门，从装置中取出板固定器。从支架中取出96孔PCR板和丢弃它。 </li><li>对于适当的保养，更换滴读者油，并根据需要清空废物容器。加入50毫升10％的漂白剂到废液瓶，以防止微生物的生长。 </li></ol> 2.4 DNA的突变分析（按制造商建议的协议） 注意:PCR-阳性和PCR阴性液滴计数，以提供绝对的quantific通货膨胀目标BRAF V600E突变的DNA以数字形式，利用数据分析软件。 <ol><li>单击设置输入有关样品，实验，和实验信息。 </li><li>在设置窗口中，加载板（filename.qlp），然后单击分析以打开和分析数据。数据分析接口被分成三个窗口:结果表，那么选择器选择和处理数据/图形显示。 </li><li>在经处理​​的数据的窗口，对每一个目标浓度数据出现在孔中板图和表列中的结果表。点击集中可视化的浓度曲线的数据。 </li></ol>

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明Ryuri哦，思恩金，和年轻的DEUG金是ABION CRO的员工。

这里，我们强调ddPCR的适用性和DNA分离自FFPE参考标准细胞系样品的特定基因突变的评估。在这项研究中，TPS自动化DNA分离方法用于其可以容易地适应，自动化，并能容纳多达48个不同的样品同时，允许较大规模的实验和较低的变异性。之一，在目前的工作中的DNA分离的局限性是，每FFPE样品是唯一的，并且将变化彼此在表面的污染物，微生物菌群，和/或人的遗传背景。在一般情况下，提取的DNA的质...

基因突变的关键调控基因的累积改变正常细胞的编程细胞样细胞的增殖，分化和存活，从而导致癌症1。在RAS-RAF-MAP激酶途径介导细胞对生长信号。致癌性的BRAF突变可以导致从驱动器中的突变的BRAF基因，这可能会导致BRAF癌蛋白成为过度活跃2。在BRAF基因的突变也导致通过MEK和ERK 3，这反过来，导致的生长因子介导的调控4-6过度细胞生长和增殖独立地过度活跃的下游信号。 有几个工具可以进行DNA突变分析，例如定量实时BRAF V600E突变的福尔马林固定石蜡包埋（FFPE）参考标准细胞株ddPCR。 ddPCR是一种基于PCR的方法，绝对定量相对于传统的定量实时PCR（qPCR的）7,8-提供更高的精度。 ddPCR还提供了更高的分辨率和精度的DNA模板稀有突变的检测，使更多的信息癌症研究和诊断的9。 ddPCR比传统的qPCR的其它优点包括其增强的灵敏度和准确度研究低模板拷贝数10-12时。这里，一个协议，用于自动提取的FFPE参考标准细胞系，接着通过确定BRAF V600E突变由ddPCR存在或不存在的DNA是证明。的软件进行数据分析，分析结果的图形表示的使用也有所说明。整个过程是相对简单且完全取决于样品进行概要的数量和常规PCR和ddPCR机器可用的数量。 以下方案描述的标准程序的BR<html> AF V600E阳性FFPE参考标准细胞系（HD598，HD593，HD617，HD273和野生型（WT））是在一个完全自动化的仪器使用组织制备系统（TPS）协议执行的。随后，分离的DNA样品用于使用ddPCR系统BRAF V600E突变的存在进行分析。进行有针对性的突变分析所有样品已异形之后和数据被加载到数据分析软件。取决于样本的数目/组研究，数据分析，可能需要从一至数小时。该方法的实验部分要求的精度在处理<a href="http://jove.unicartagenaproxy.elogim.com/science-education/5096/isolating-nucleic-acids-from-yeast" title="从酵母中分离核酸，一个朱庇特的科学教育视频解释有关RNA的背景下更多">的DNA</a>和rological移液器和移液器，一朱庇特科学教育视频解释有关移液的上下文中了解更多"&gt;移液到96孔板中，数据分析是用软件来执行时。

<table><tbody><tr><td>Hamilton MICROLAB STARlet IVD instrument</td><td>Siemens</td><td>10701001</td><td>Automated DNA isolation instrument</td></tr><tr><td>QX200 Droplet Generator&amp;nbsp;</td><td>Bio-Rad</td><td>772BR1119</td><td></td></tr><tr><td>QX200 Droplet Reader</td><td>Bio-Rad</td><td>771BR1497</td><td></td></tr><tr><td>Conventional PCR machine capable of ramp-time adjustment</td><td></td><td>621BR17718</td><td></td></tr><tr><td>PX1 PCR plate sealer</td><td>Bio-Rad</td><td>770BR1575</td><td></td></tr><tr><td>QuantaSoft software</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>DNA isolation kit&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>VERSANT Tissue Preparation Reagents Box 1&amp;nbsp;</td><td>Siemens</td><td>10632398</td><td></td></tr><tr><td>VERSANT Tissue Preparation Reagents Box 1&amp;nbsp;</td><td>Siemens</td><td>10632399</td><td></td></tr><tr><td>CO-RE tips</td><td>Siemens</td><td></td><td></td></tr><tr><td>ddPCR mutation analysis</td><td></td><td></td><td></td></tr><tr><td>ddPCR Supermix&amp;nbsp;</td><td>Bio-Rad&amp;nbsp;</td><td>BR186-3010</td><td>2X concentration</td></tr><tr><td>DG8 cartridge&amp;nbsp;</td><td>Bio-Rad&amp;nbsp;</td><td>BR186-4008</td><td></td></tr><tr><td>Droplet Generator oil</td><td>Bio-Rad&amp;nbsp;</td><td>BR-186-3005</td><td></td></tr><tr><td>Gasket</td><td>Bio-Rad&amp;nbsp;</td><td>BR186-3006</td><td></td></tr><tr><td>Droplet reader oil</td><td>Bio-Rad&amp;nbsp;</td><td>BR-186-3004</td><td></td></tr></tbody></table>

employing digital droplet pcr to detect braf v600e mutations in formalin-fixed paraffin-embedded reference standard cell lines

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is partitioned into ~20,000 nano-sized, water-in-oil droplets, and PCR amplification occurs in individual droplets. The ddPCR approach is in identifying sequence mutations, copy number alterations, and select structural rearrangements involving targeted genes. Here, we demonstrate the use of ddPCR as a powerful technique for precisely quantitating rare BRAF V600E mutations in FFPE reference standard cell lines, which is helpful in identifying individuals with cancer. In conclusion, ddPCR technique offers the potential to precisely profile the specific rare mutations in different genes in various types of FFPE samples.

对于我们的ddPCR分析中，我们研究了BRAF V600E突变FFPE参考标准的细胞系。液滴阅读器连接到笔记本电脑上运行的数据分析软件。每个单独的微滴荧光幅度来作为阳性或阴性上定义。由制造商提供的软件还允许输入用户定义的截止定义的正和负的液滴之间的阈值。样品中的正和负液滴的数量用于计算在拷贝/微升术语靶的浓度。 荧光检测和加工成二维散点图显示，定制软件...

Watch this Scientific Journal Video about Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines at JoVE.com

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

该视频的目标是展示如何执行从福尔马林固定的石蜡包埋（FFPE）的参考标准细胞系和数字液滴的PCR（ddPCR）分析的自动化DNA提取，以检测稀有突变在临床环境中。 FFPE样品中检测突变表明ddPCR的FFPE样品中的临床应用。

采用数字液滴PCR技术检测BRAF V600E突变的福尔马林固定石蜡包埋的参考标准细胞系

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is ...

employing-digital-droplet-pcr-to-detect-braf-v600e-mutations-formalin

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

13.2K 次观看.  Seoul National University. 该视频的目标是展示如何执行从福尔马林固定的石蜡包埋（FFPE）的参考标准细胞系和数字液滴的PCR（ddPCR）分析的自动化DNA提取，以检测稀有突变在临床环境中。 FFPE样品中检测突变表明ddPCR的FFPE样品中的临床应用。 

视频: Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer. Our workflow includes a pathologist review of the biopsy to ensure there is adequate amount of tumor for the assay followed by customized DNA extraction is performed on the specimen. Quality control of the specimen includes steps for quantity, quality and integrity and only after the extracted DNA passes these metrics an amplicon library is generated and sequenced. The resulting data is analyzed through an in-house bioinformatics pipeline and the variants are reviewed and interpreted for pathogenicity. Here we provide a snapshot of the utility of each panel using two clinical cases to provide insight into how a well-designed NGS workflow can contribute to optimizing clinical outcomes.

This manuscript describes clinical protocols for two next-generation sequencing panels. One panel interrogates hematologic malignancies while the other panel targets genes commonly mutated in solid tumors. Molecular classification of driver mutations in human malignancies offers valuable prognostic and predictive information.

新一代测序为可操作的突变在固体和液体瘤检测

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific ...

科研

癌症研究

Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations

Accurate detection and identification of low frequency mutations can be problematic when assessing residual disease after therapy, screening for emerging resistance mutations during therapy, or when patients have few circulating tumor cells. Wild-type blocking PCR followed by sequencing analysis offers high sensitivity, flexibility, and simplicity as a methodology for detecting these low frequency mutations. By adding a custom designed locked nucleic acid oligonucleotide to a new or previously established conventional PCR based sequencing assay, sensitivities of approximately 1 mutant allele in a background of 1,000 WT alleles can be achieved (1:1,000). Sequencing artifacts associated with deamination events commonly found in formalin fixed paraffin embedded tissues can be partially remedied by the use of uracil DNA glycosylase during extraction steps. The optimized protocol here is specific for detecting MYD88 mutation, but can serve as a template to design any WTB-PCR assay. Advantages of the WTB-PCR assay over other commonly utilized assays for the detection of low frequency mutations including allele specific PCR and real-time quantitative PCR include fewer occurrences of false positives, greater flexibility and ease of implementation, and the ability to detect both known and unknown mutations.

Wild-type blocking PCR followed by direct sequencing offers a highly sensitive method of detection for low frequency somatic mutations in a variety of sample types.

野生类型阻止PCR结合直接测序作为低频体细胞突变的检测的高灵敏度的方法

Accurate detection and identification of low frequency mutations can be problematic when assessing residual disease after therapy, screening for emerging ...

遗传学

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

用触印细胞学方法快速获得临床病理标本中高质量肿瘤 DNA

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

单液滴数字聚合酶链反应对热点地区突变的综合和同步检测

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

患者生物流体中肿瘤相关循环DNA的检测与监测

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Naked-Eye Detection of Rare Point Mutations in DNA

The protocol describes a naked-eye colorimetric test for the detection of somatic point mutations in an excess of wild type DNA. The future foreseen application of the method is the identification of rare mutations in circulating cell-free DNA from liquid biopsies, with a relevance in cancer diagnostics and stratification of oncological patients for personalized therapy. As a proof of concept, the test has been designed to detect the BRAFV600E mutation in the BRAF gene, which is important to identify the sub-group of melanoma patients that can benefit from targeted therapies with BRAF inhibitors. However, this colorimetric test can be easily generalized to other somatic mutations of clinical relevance due to the use of universal detection probes, thus providing strong potential in oncological diagnostics.
The test detects 0.5% of BRAFV600E in an excess of BRAFWT DNA, which matches the sensitivity of some commercial instrumental assays. Such sensitivity is clinically relevant for diagnostic purposes, allowing the early identification of drug-sensitive patients. In contrast to commercial assays based on real-time PCR, this test requires minimal instrumentation and processing, as it can be performed on DNA amplified with a standard PCR (or isothermal techniques) and provides a naked-eye readout with a one-tube reaction of a few steps in only one hour. At present, the test has been used only on synthetic DNA samples. However, the latter have been designed to mimic a real sample amplified from circulating cell-free DNA, to favor the translation of the test to clinical diagnostics.

This protocol permits the naked-eye identification of point mutated DNA in a 200-fold excess of wild type DNA molecules, by exploiting gold nanoparticles and paramagnetic microparticles.

The protocol describes a naked-eye colorimetric test for the detection of somatic point mutations in an excess of wild type DNA. The future foreseen ...

生物化学

增强癌症组织样本中致癌驱动因素的 FISH 分析

Robust Detection of Gene Amplification in Formalin-Fixed Paraffin-Embedded Samples by Fluorescence In Situ Hybridization

Focal gene amplification, such as extrachromosomal DNA (ecDNA), plays an important role in cancer development and therapy resistance. While sequencing-based methodologies enable an unbiased identification of ecDNA, cytogenetic-based techniques, such as fluorescence in situ hybridization (FISH), remain time and cost-effective for identifying ecDNA in clinical specimens. The application of FISH in formalin-fixed paraffin-embedded (FFPE) tissue samples offers a unique avenue for detecting amplified genes, particularly when viable specimens are not available for karyotype examination. However, there is a lack of consensus procedures for this technique. This protocol provides comprehensive, fully optimized, step-by-step instructions for conducting FISH to detect gene amplification, including ecDNA, in FFPE tissue samples which present unique challenges that this protocol aims to overcome and standardize. By following this protocol, researchers can reproducibly acquire high-quality imaging data to assess gene amplification.

作者聚焦：FISH 作为癌症标本中精确基因扩增评估的工具

This protocol provides a reproducible method to visualize gene amplification in formalin-fixed paraffin-embedded (FFPE) tissue specimens.

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

Focal gene amplification, such as extrachromosomal DNA (ecDNA), plays an important role in cancer development and therapy resistance. While ...

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using bioinformatics pipelines (&#34;dry bench&#34;). Both elements are essential to produce accurate and reliable results, which are particularly critical for clinical laboratories. Targeted NGS technologies have increasingly found favor in oncology applications to help advance precision medicine objectives, yet the methods often involve disconnected and variable wet and dry bench workflows and uncoordinated reagent sets. In this report, we describe a method for sequencing challenging cancer specimens with a 21-gene panel as an example of a comprehensive targeted NGS system. The system integrates functional DNA quantification and qualification, single-tube multiplexed PCR enrichment, and library purification and normalization using analytically-verified, single-source reagents with a standalone bioinformatics suite. As a result, accurate variant calls from low-quality and low-quantity formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration (FNA) tumor biopsies can be achieved. The method can routinely assess cancer-associated variants from an input of 400 amplifiable DNA copies, and is modular in design to accommodate new gene content. Two different types of analytically-defined controls provide quality assurance and help safeguard call accuracy with clinically-relevant samples. A flexible &#34;tag&#34; PCR step embeds platform-specific adaptors and index codes to allow sample barcoding and compatibility with common benchtop NGS instruments. Importantly, the protocol is streamlined and can produce 24 sequence-ready libraries in a single day. Finally, the approach links wet and dry bench processes by incorporating pre-analytical sample quality control results directly into the variant calling algorithms to improve mutation detection accuracy and differentiate false-negative and indeterminate calls. This targeted NGS method uses advances in both wetware and software to achieve high-depth, multiplexed sequencing and sensitive analysis of heterogeneous cancer samples for diagnostic applications.

An integrated system for targeted next-generation sequencing of oncology specimens is described. This cross-platform system is optimized for low-quality and low-quantity tumor biopsies, accommodates low DNA inputs, includes well-characterized multi-variant controls, and features a novel variant caller that is informed by quantitative pre-analytical quality control measures.

低质量和低数量肿瘤活检丰枯台过程的集成优化了有针对性的下一代测序

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using ...

采用数字液滴PCR技术检测BRAF V600E突变的福尔马林固定石蜡包埋的参考标准细胞系

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此视频中的章节

新一代测序为可操作的突变在固体和液体瘤检测

野生类型阻止PCR结合直接测序作为低频体细胞突变的检测的高灵敏度的方法

用触印细胞学方法快速获得临床病理标本中高质量肿瘤 DNA

单液滴数字聚合酶链反应对热点地区突变的综合和同步检测

患者生物流体中肿瘤相关循环DNA的检测与监测

Naked-Eye Detection of Rare Point Mutations in DNA

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

低质量和低数量肿瘤活检丰枯台过程的集成优化了有针对性的下一代测序