Name: digital polymerase chain reaction assay for the genetic variation in a sporadic familial adenomatous polyposis patient using the chip-in-a-tube format
Uploaded: 2018-08-20T14:30:00.000+00:00
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Description: Watch this Scientific Journal Video about Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format at JoVE

这项研究得到了日本促进科学学会 (15K08397) 对智明 Kahyo 的资助, 以及日本医学研究与发展署 (阿曼德) 的赠款 (927960719 号),为探索性研究提供的补助 (16K15256), 为促进全国大学管理费用赠款改革特别预算的相关项目支出 (1019253 号), 以及吸烟研究基金会, 以黑田东彦 Sugimura。作者感谢 Iwaizumi 博士和 Kurachi 博士的临床支持。出资人在编写手稿方面没有任何作用。图 3、图 4和表 4是根据 Kahyo等的文章改编和转载的。10, 征得 Elsevier 的许可。

研究设计由滨松大学医学院 (G-260-4) 的机构审查委员会批准。从病人和他的父母那里获得书面知情同意。
1. 基因组 DNA 的质量控制
注: 利用已建立的基于硅膜的 dna 纯化方法, 从外周血中提取基因组 dna (gDNA)。在下面的程序之前, 使用分光光度法测定 gDNA 的浓度。
<ol><li>在10–100µL 的浓度下制备 gDNA 样品。</li>
	<li>将10µL 的 DNA 样本缓冲区添加到8管带上。</li>
	<li>将1µL 的 DNA 阶梯或 gDNA 添加到试管中。使用微板块附件将混合物涡流1分钟。</li>
	<li>将管、凝胶装置和吸管贴入电泳仪 (材料表), 按 "开始" 按钮开始运行。 注: 电泳系统将自动操作, 一次点击开始按钮。</li>
	<li>确认在 electropherograms 上正确分配了 DNA 样本缓冲区中包含的较低标记。如果分配不正确, 请手动在软件的 "Electroherogram 模式" 中指定标记。 注: DNA 完整性号 (DIN) 将自动计算。DIN 值来自 DNA 完整性。高和低 DIN 值表示高度原封和强烈退化的 gDNA, 分别。</li>
	<li>指定软件 "区域模式" 中的 gDNA (> 200 bp) 的大小区域, 以自动计算 gDNA (> 200 bp) 的浓度。</li>
</ol>2. 底漆和探头设计
<ol><li>在以下条件下使用任何开放的访问工具计算熔化温度 (Tm) 值: 10–25基, 50 毫米 Na+/K+, 0.80 毫米 dNTPs, 3 毫米毫克2 + (材料表)。设计正向和反向引物, 放大含有目标等位基因的基因组区域, 其 Tm值约60摄氏度, 扩增子长度在 100–300 bp。 注: 请参阅表 1中的寡核苷酸浓度。</li>
	<li>根据以下条件设计了用于参考和变异等位基因的锁定核酸探针: (i) 1–6 LNAs 存在于每个探针中;(ii) 每个探针的 5 '-和 3 '-总站上分别存在荧光染料和淬火;(iii) 匹配和不匹配探针的 Tm值之间的区别是 > 10 °c;(iv) 匹配和不匹配探针的 Tm值分别高于引物 (例如, 向前底漆: 60.8 °c, 反向底漆: 59.8 °c, 参考等位基因探针 (匹配/不匹配): 62.6/45.3 °c, 变体等位基因探针 (匹配/不匹配): 61.7/50.5 °c]。Tm值是使用用于步骤2.1 的开放访问工具计算的。</li>
	<li>确保所设计的引物和探针不包含单核苷酸多态性, 频率为 > 0.1%, 由数据库确定 [例如, 单核苷酸态性数据库 (dbSNP)]。</li>
</ol>3. 数字 PCR
<ol><li>用 TE 缓冲器在表 1中描述的浓度中准备底漆、探针和 gDNA 库存解决方案, 并将它们存储在20–4°c。</li>
	<li>
将试剂在室温下混合, 总容积为15µL 到最后浓度, 如表1所述。
	<ol>
<li>在无模板控制 (NTC) 中添加3µL 的蒸馏水, 而不是 gDNA。</li>
		<li>准备3.5x 的 PCR 混合物的数量三个, 以解释吹打错误。</li>
	</ol>
</li>
	<li>将 PCR 混合物上下吸管混合。</li>
	<li>将加载平台设置在8管带上的芯片上, 并将其设置在自动加载机上。确保芯片与加载平台之间有联系。</li>
	<li>在平台上安装加载滑块, 并将滑块与装载机的塞子保持在一起。</li>
	<li>吸管15µL 的 PCR 混合物在平台附近的滑块尖端 (图 1A)。</li>
	<li>按加载程序的按钮 (大约1分钟) 运行加载程序。 注意: 如果在平台上保持少量 PCR 混合物, 这不是问题。可以按顺序重新运行加载程序。</li>
	<li>在密封增强剂的侧面槽中, 将带 PCR 混合物的芯片装入管内。推滑盖和顶盖的边缘, 以不打破芯片 (图 1B)。</li>
	<li>运行密封增强器 (约2分钟)。按顺序重新运行密封增强器1分钟, 如果液体的水坑仍然可见, 因为不完整的密封导致对正面信号的交叉污染 (图 1C和1D)。</li>
	<li>添加230µL 的密封液, 一个油基试剂, 到管。 注: 芯片现在应该浸入液体中。</li>
	<li>在热循环仪中设置管。运行热循环仪如表 2所述 (约2小时)。如果正分区分布不均匀 (图 1E), 则调整 PCR 的温度或持续时间。 注: 建议将坡道速度设置为约1摄氏度/秒。</li>
	<li>将管放在热循环仪中至少15分钟, 以减少基线噪音。 注意: 管子也可以在一夜之间被留下。甚至在第二天也检测到了荧光信号。</li>
	<li>在检测夹具上设置管, 将6毫升蒸馏水倒入夹具中。如果气泡在管子内外可见, 请清除它们 (图 1F和1G)。</li>
	<li>将夹具加载到检测器中, 然后在选择荧光、实验和示例/NTC 标签 (材料表) 后, 单击软件的 "运行" 按钮即可运行它。 注: 对八个具有默认强度的样本进行双色检测需要大约 4-5 分钟。</li>
	<li>确定位置图、直方图和2D 散点图。 注: iIf 的荧光强度的负面分区是高, 调整强度, 点击设置按钮的软件, 使其属于30–70范围内。</li>
	<li>使用以下公式计算变体等位基因分数 (VAF): <img alt="Equation 1" src="/files/ftp_upload/58199/58199eq1.jpg"> 在这里, Cv 和 Cr 分别是变体和参考等位基因的拷贝数。 注: 在基于泊松统计的软件程序中自动计算它们。</li>
</ol>4. PCR 产品的收集
<ol><li>从管子上取下密封液。</li>
	<li>将100µL 的 TE 缓冲器添加到管中。漩涡强烈为三十年代和简要地离心机管子。</li>
	<li>将 TE 溶液转移到另一管, 加入30µL 3 米醋酸钠和200µL 乙醇。</li>
	<li>冷却溶液在-20 摄氏度过夜。</li>
	<li>离心溶液为30分钟在 1.6万 x g和去除上清。</li>
	<li>添加300µL 80% 乙醇的管和涡。</li>
	<li>离心溶液为15分钟在 1.6万 x g和去除上清。使沉淀的空气干燥5分钟。</li>
	<li>在空气干燥后1–5µL 中溶解沉淀。</li>
	<li>如有必要, 可参照步骤 1, 使用电泳仪器评估产品尺寸。将溶液的1µL 对凝胶装置进行高灵敏度 (材料表)。</li>
</ol>

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作者没有什么可透露的。

DIN 值通常用于评估被破坏的 DNA (例如, gDNA 从福尔马林固定石蜡嵌入组织) 在定量或排序之前, 因为 gDNA 的高级退化可能导致低质量数据。因此, DIN 评估已成为人类 gDNA11质量控制的重要一步。因为在代表性实验使用的 gDNA 在它从血液提取以后被存放了在4°c 为4–11年, 它的 DIN 价值被确定了为质量控制在 dPCR 试验之前。如果材料被怀疑损坏, 则特别推荐此步骤。gDNA 的浓度也由电泳法测定。由于 dPCR 的动态范围不像 qPCR 由于分区的局限性而宽, gDNA 浓度的测量是成功 dPCR 检测的重要步骤。由于输入 DNA 的数量与 dPCR 测定中变异和参考等位基因的总拷贝数相关 (R-平方 = 0.935;表 4), 电泳对测定 dPCR 测定前的 gDNA 浓度有很重要的意义。
加载和密封过程是成功结果的重要步骤。确定在平台上适当安装 PCR 混合物并确保芯片与平台之间的接触是至关重要的 (图 1A)。从滑块中的 PCR 混合物的凸出可能导致芯片上的分布无效.确认在位置图上的正和负分区的分布也需要精确分析, 因为在泊松统计中假定 DNA 模板被随机分成1室。在代表性的结果中, 在整个芯片中分布有正的分区 (图 3)。这一结果符合泊松统计的要求, 反映了加载和密封程序是有效的。当在密封增强器中设置管时, 如果顶盖的中心部分被推得太强 (图 1B), 芯片可能会断裂。要避免这种情况, 请轻轻推顶盖的边缘。如果存在正分区的人工簇 (图 1D), 则由于分区之间的交叉污染, 复制编号可能会过高估计。如果表面上可见一滩液体 (如图 1C) (例如, 通过依次重新运行密封增强剂1分钟), 则应改进密封过程。如果正分区分布不均匀 (图 1E), 则由于密封液和水的放大或污染不足, 复制数可能被低估。pcr 条件应在这种情况下调整 [例如, 通过调整温度或 pcr 的持续时间 (步骤3.11 的协议), 或确保密封流体和水倒入夹具是干净的]。从浸泡在蒸馏水中的8管条中检测出荧光信号。如果气泡附着在管表面, 就有可能干扰信号检测 (图 1F)。因此, 气泡应该使用一个工具, 如细吸管提示清除。此外, 空气气泡可能形成的管道内, 当他们设置在夹具 (图 1G)。如果管子内的气泡足够大, 足以覆盖芯片, 它们也应该被清除。气泡可能会清除, 如果他们在室温下离开几分钟。
在 NTC 中可能检测到一些积极的分区。为了避免 DNA 模板的污染, 保持工作台的清洁, 如果可能的话, 用风扇过滤单元制造一个干净的空间。如果怀疑 DNA 模板的污染, 彻底清除空间和/或丢弃使用过的试剂。相比之下, 如果在 gDNA 存在时未检测到参考等位基因的阳性分区, 请重新确认 gDNA、底漆和探针的浓度。值得注意的是, 与常规 PCR (表 1)12相比, 引物在代表性实验中使用的浓度较低。在常规 PCR 中, 确定斜坡速率是非常重要的, 这种速度很少改变。
是什么使 dPCR 与芯片在一管格式独特的是分割 PCR 混合物内的通用8管条8,13。该系统不需要将分区腔转移到 PCR 管中, 从而降低了污染风险。还有一个优势, 在 dPCR 系统与芯片在一管格式;它需要 < 4 小时完成96化验, 在该 dPCR 系统, 而它需要至少5小时完成相同数量的化验, 以液滴为基础的 dPCR14。此外, 通用8管带允许使用传统的热循环仪, 不像另一个基于芯片的 dPCR15, 这需要一个热循环仪与扁平块。此外, 常规 PCR 的积累知识和试剂也可应用于 dPCR 系统。这将有助于扩大 dPCR 的应用。
应该指出的是, dPCR 有几个限制。在 dPCR 中, 与其他 dPCR 平台相比, 芯片的分区数相对较低。对芯片设备的进一步改进将需要增加动态范围, 这取决于分区的数量。由于通用管的内部空间有限, 因此需要对芯片设备进行突破性设计, 以增加分区数量。未来整个 dPCR 程序的自动化将大大减少人为错误, 从而产生更可靠的结果。由于其高吞吐量和高灵敏度的性质, dPCR 与芯片内管的格式有望被应用于处理一些样品的液体活检和环境 DNA。

定量 PCR (qPCR) 经常用于量化人类遗传变异, 包括单核苷酸变异 (SNVs) 和 DNA 拷贝数变异 (CNVs)。在 qPCR 中, 在每管中进行聚合酶反应的方式与常规终点 PCR 方法相同, 在每个热循环后从管中获得放大信号。相比之下, 在 dPCR, 这意味着终点 dPCR 在本报告中, PCR 混合物被加载到许多微观室, 称为分区, 其中 DNA 模板是存在或缺席的限制稀释, 和每一个包含 PCR 混合物的分区被评估为完全 PCR 后阴性或阳性。虽然很容易估计在负分区中没有 dna 模板, 但目前尚不清楚阳性分区中有多少个 dna 模板拷贝。因此, 在正分区中的 DNA 模板数根据泊松分布估计, 使用来自负分区1的计数。与 qPCR 相比, dPCR 成本更高, 动态范围更小, 但是这种方法能够实现绝对量化, 并提供更高的灵敏度和更精确的2。
dPCR 的主要应用之一是对下一代测序识别的变种进行验证。特别是在罕见变型的情况下, 这意味着低变型分数, 验证是至关重要的, 因为排序错误可能发生在3。虽然 qPCR 是验证 SNVs 和 CNVs 的有用工具, 但与 dPCR 相比, 它们的灵敏度较低。因此, dPCR 技术对处理稀有变种的遗传研究是有需求的。最近, 经液体活检发现的罕见变种与癌症的特点, 如耐药性, 已成为分子诊断和治疗的热门话题4。dPCR 技术似乎适合于液体活检研究, 预计在不久的将来将有重要的临床应用, 尽管有关动态范围和成本的改进仍需实施。
商业可用的 dPCR 技术大致可以分为液滴和基于芯片的平台;不同的是 DNA 模板是如何划分5,6,7。在 dPCR 与管状芯片的格式, PCR 混合物由毛细管作用分配到芯片上的分区。芯片是建立在八管条, 许多实验室工作人员将已经熟悉, 因此, 八样品可以处理一次8。在阅读步骤中, 通过检测芯片的整个图像而不是每个分区来处理96个样本需要 < 1 小时。由于 dPCR 与其他 dPCR 系统相比具有较高的吞吐量, 因此, 可用性和生产率是其用户的主要优势。
在本报告中, 以零星家族性腺瘤性息肉病患者的APC基因体细胞嵌合体为典型病例, 并比较 dPCR 和结果。本报告的主要目的是要清楚地量化一个单一核苷酸变体使用 dPCR 与芯片在一管格式。我们希望这份报告能帮助感兴趣的研究人员为自己的工作采用 dPCR 平台。

<table><tbody><tr><td>QIAamp DNA Blood Maxi Kit</td><td>Qiagen</td><td>51194</td><td>Before Protocol 1: Extraction of genomic DNA</td></tr><tr><td>NanoDrop 1000</td><td>ThermoFisher SCIENTIFIC</td><td>ND-8000</td><td>Before Protocol 1: Spectrophotometer</td></tr><tr><td>8-strip tube</td><td>Agilent Technologies</td><td>401428</td><td>Protocol 1</td></tr><tr><td>Genomic DNA sample buffer</td><td>Agilent Technologies</td><td>5067-5366</td><td>Protocol 1:&lt;br/&gt; A component of Genomic DNA Screen Tape Assay</td></tr><tr><td>DNA ladder</td><td>Agilent Technologies</td><td>5067-5366</td><td>Protocol 1:&lt;br/&gt; A component of Genomic DNA Screen Tape Assay</td></tr><tr><td>MS3 Basic Small Shaker</td><td>IKA</td><td>3617000</td><td>Protocol 1: Vortex mixer</td></tr><tr><td>Genomic DNA ScreenTape</td><td>Agilent Technologies</td><td>5067-5365</td><td>Protocol 1: Gel device</td></tr><tr><td>2200 TapeStation system</td><td>Agilent Technologies</td><td>G2965AA</td><td>Protocol 1: Electrophoresis instrument</td></tr><tr><td>TapeStation Analysis Software</td><td>Agilent Technologies</td><td>Bundled with G2965AA</td><td>Protocol 1: Analysis software</td></tr><tr><td>DNA oligo primers</td><td>IDT</td><td>Custom order</td><td>Protocol 2</td></tr><tr><td>LNA probes</td><td>IDT</td><td>Custom order</td><td>Protocol 2</td></tr><tr><td>Software tool</td><td>IDT</td><td>Web site: http://biophysics.idtdna.com/</td><td>Protocol 2</td></tr><tr><td>dbSNP database</td><td>NCBI</td><td>Web site: https://www.ncbi.nlm.nih.gov/projects/SNP/</td><td>Protocol 2</td></tr><tr><td>TE buffer</td><td>ThermoFisher SCIENTIFIC</td><td>12090015</td><td>Protocol 3</td></tr><tr><td>DNase/RNase-Free Distilled Water</td><td>ThermoFisher SCIENTIFIC</td><td>10977015</td><td>Protocol 3 (Table 1)</td></tr><tr><td>Clarity Digital PCR Probe Mastermix</td><td>JN Medsys</td><td>12013</td><td>Protocol 3 (Table 1): 2xPCR Master Mix&lt;br/&gt; A component of #10011</td></tr><tr><td>Clarity Sealing Fluid</td><td>JN Medsys</td><td>12005</td><td>Protocol 3: Sealing fluid&lt;br/&gt; A component of #10011</td></tr><tr><td>Clarity JN Solution</td><td>JN Medsys</td><td>12006</td><td>Protocol 3 (Table 1): 20xdPCR solution&lt;br/&gt; A component of #10011</td></tr><tr><td>Clarity Tube-strip</td><td>JN Medsys</td><td>12007</td><td>Protocol 3: Chip-in-a-tube&lt;br/&gt; A component of #10011</td></tr><tr><td>Clarity Sample Loading Kit</td><td>JN Medsys</td><td>12008</td><td>Protocol 3: Loading platform and slider&lt;br/&gt; A component of #10011</td></tr><tr><td>Clarity Auto Loader</td><td>JN Medsys</td><td>11002</td><td>Protocol 3: Auto loader&lt;br/&gt; A component of #10001</td></tr><tr><td>Clarity Sealing Enhancer</td><td>JN Medsys</td><td>11003</td><td>Protocol 3: Sealing enhancer&lt;br/&gt; A component of #10001</td></tr><tr><td>Clarity Reader</td><td>JN Medsys</td><td>11004</td><td>Protocol 3: Reader&lt;br/&gt; A component of #10001</td></tr><tr><td>Life Eco Thermal Cycler</td><td>Bioer Technology</td><td>TC-96GHbC</td><td>Protocol 3: Thermal cycler</td></tr><tr><td>Clarity Software</td><td>JN Medsys</td><td>Bundled with #10001</td><td>Protocol 3: Analysis software</td></tr><tr><td>High Sensitivity D1000 screen tape</td><td>Agilent Technologies</td><td>5067-5584</td><td>Protocol 4: Gel device for high sensitivity</td></tr></tbody></table>

digital polymerase chain reaction assay for the genetic variation in a sporadic familial adenomatous polyposis patient using the chip-in-a-tube format

对人类遗传变异的定量分析对于了解肿瘤等严重疾病的分子特征至关重要。由于数字聚合酶链反应 (PCR) 能够精确量化 DNA 拷贝数变体, 它们正成为检测稀有遗传变异的重要工具, 如耐药性突变。预计在不久的将来, 使用数字 PCR (dPCR) 的分子诊断将在临床实践中得到应用;因此, 如何有效地进行 dPCR 的人类遗传物质是一个热门话题。在这里, 我们介绍了一种检测腺瘤型息肉样大肠杆菌(APC) 体细胞嵌合体的方法, 使用 dPCR 与芯片内管格式, 这使得八 dPCR 反应同时进行。在填充和密封的反应混合物在芯片上应注意。本文演示如何避免对正分区的过度和低估。此外, 我们提出了一个简单的程序, 从芯片上的分区收集 dPCR 产品, 然后可以用来确认具体放大。我们希望这种方法的报告将有助于促进 dPCR 与芯片内管方法在遗传学研究。

根据上述的程序, 我们回顾了一个有零星家族性腺瘤性息肉病患者的APC基因的体细胞嵌合体。在先前的一项研究中, 834 + 2T > c 的变异是在病人身上发现的, 但在他们的父母中却没有, 使用了桑格测序和9。表 3显示了代表性结果中使用的引物和探头的设计。基因组 DNA 从先证者、父母和六健康捐献者的血液中提取。九 gDNA 样品的 DIN 值范围到 6.7–7.9 (图 2和表 4)。分别用家族和十六进制作为 C 和 T 等位基因的荧光染料。位置图上的绿色和黄色斑点分别代表了两个正则和六角正的分块 (数字 3A)。蓝色斑点代表了两个家族和十六进制的负分区。黑色背景对应于未添加反应混合物的分区。在先证者中发现了 c 和 T 等位基因的许多阳性分区, 而在先证者的父亲或 NTC 中检测到 c 等位基因的阳性分区很少, 而后者则未发现 T 等位基因的阳性分区。在二维 (2 维) 散点图上, C 和 T 等位基因之间的信号分离是明确的, 而十六进制和家族的最正信号是相互排斥的 (图 3B)。VAF 的先证者是 13.2%, 这是类似于确定使用的 (12.7%)(表 4)。先证者的父母和健康的捐赠者的 VAFs 是 < 0.1%。APC c. 834 + 2T > c 突变的检测极限被估计为 0.3%, 使用3x 的重复测量的标准偏差与总 gDNA10的10–11。
834 + 2T > c 扩增子的 dPCR 检测量预测为 123 bp。为了确认 dPCR 产品被放大为一个单一的波段, dPCR 产品收集从测定芯片。使用电泳, 一个单一的波段被清楚地检测, 和产品的大小是一致的预测 (图 4)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58199/58199fig1.jpg"> 图 1: dPCR 用管状芯片的格式进行检测的注意事项.(A) 本小组展示如何在自动加载机中设置8管条。(B) 本小组显示碎薯条。(C) 此面板显示了在 (i) 加载和 (ii) 密封后芯片表面的情况。(iii) 在密封不完全的情况下, 可看到液体的水坑。(D) 本小组显示在交叉污染情况下的位置图。(E) 本小组在分配不均的情况下显示了立场图。(F) 本小组展示了管表面气泡如何浸入水中。(G) 此面板显示管内的气泡。在整个图中, 黑白箭头分别表示破碎的芯片碎片和气泡。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58199/58199fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58199/58199fig2.jpg"> 图 2: 代表血液 gDNA 的 electropherograms.Electropherograms 的血液 gDNA 与 DIN 值6.7 和7.9 分别显示在上部和底部的面板上。星号表示较低的标记。DIN: DNA 完整性数字。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58199/58199fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58199/58199fig3.jpg"> 图 3: APC体细胞嵌合体的代表性结果使用 dPCR 与芯片在 a 管格式.(a) scatterplots 的位置图和 (B) 无模板控制 (NTC)、先证者和父亲的情况显示在这里。十六进制和 FAM 分别对应于参考和变异的等位基因。垂直和水平线分别表示十六进制和 FAM 强度的阈值。这个数字已经从 Kahyo等。10.<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58199/58199fig3large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58199/58199fig4.jpg"> 图 4: 从测定芯片中收集 dPCR 产品.收集和浓缩的 dPCR 产品均受到电泳。预测的目标大小为 123 bp。这个数字已经从 Kahyo等。10. NTC: 无模板控制。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58199/58199fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>试剂</td>
			<td>浓度 的库存解决方案</td>
			<td>容量 (ul)</td>
			<td>最终浓度</td>
			<td>推荐 最终浓度</td>
		</tr>
<tr>
<td>DNase/RNase 无水蒸馏水</td>
			<td>-</td>
			<td>1.75</td>
			<td>-</td>
			<td>-</td>
		</tr>
<tr>
<td>2x PCR 大师组合</td>
			<td>-</td>
			<td>7。5</td>
			<td>-</td>
			<td>-</td>
		</tr>
<tr>
<td>主要等位基因的低噪声探针</td>
			<td>2微米</td>
			<td>0。5</td>
			<td>66.7 毫微米</td>
			<td>3.33–100 nM</td>
		</tr>
<tr>
<td>小等位基因的低噪声探头</td>
			<td>2微米</td>
			<td>0。5</td>
			<td>66.7 毫微米</td>
			<td>3.33–100 nM</td>
		</tr>
<tr>
<td>前进底漆</td>
			<td>1微米</td>
			<td>0。5</td>
			<td>33.3 毫微米</td>
			<td>3.33–50.0 nM</td>
		</tr>
<tr>
<td>反向底漆</td>
			<td>1微米</td>
			<td>0。5</td>
			<td>33.3 毫微米</td>
			<td>3.33–50.0 nM</td>
		</tr>
<tr>
<td>20x dPCR 解决方案</td>
			<td>-</td>
			<td>0.75</td>
			<td>-</td>
			<td>-</td>
		</tr>
<tr>
<td>gDNA</td>
			<td>3.33 ng/ul</td>
			<td>3</td>
			<td>666页/ul</td>
			<td>20 pg/μL–2,000 pg/ul1
</td>
		</tr>
</tbody></table>表 1: 用于 dPCR 化验的试剂.1取决于预期的精度和灵敏度。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>步</td>
			<td>温度</td>
			<td>时间</td>
			<td>周期</td>
		</tr>
<tr>
<td>初始变性</td>
			<td>95°c</td>
			<td>5米</td>
			<td>1</td>
		</tr>
<tr>
<td>变性 退火和延伸</td>
			<td>95°c 58°c</td>
			<td>五十年代 九十年代</td>
			<td>42</td>
		</tr>
<tr>
<td>最终扩展名</td>
			<td>70°c</td>
			<td>5米</td>
			<td>1</td>
		</tr>
</tbody></table>表 2: 热循环仪条件。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>名字</td>
			<td>序列1</td>
			<td>Tm值2</td>
		</tr>
<tr>
<td>前进底漆</td>
			<td>5 '-GGTCAAGGAGTGGGAGAAATC-3′</td>
			<td>60.8 °c</td>
		</tr>
<tr>
<td>反向底漆</td>
			<td>5 '-TCTTAGAACCATCTTGCTTCATACT-3′</td>
			<td>59.8 °c</td>
		</tr>
<tr>
<td>T 等位基因的低噪声放大探针</td>
			<td>5 '-十六进制-ATTT [A] CCTGACCA-IBFQ-3 '</td>
			<td>匹配: 62.6 °c 不匹配: 45.3 °c</td>
		</tr>
<tr>
<td>C 等位基因的低噪声探针</td>
			<td>5 '-TTT [G] CCTGACC-IBFQ-3 '</td>
			<td>匹配: 61.7 °c 不匹配: 50.5 °c</td>
		</tr>
</tbody></table>表 3: 引物和探头的设计.1带下划线和括号内的字母分别是低噪声放大和目标变量。2根据协议的步骤2计算。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>3</td>
			<td colspan="4">dPCR4</td>
		</tr>
<tr>
<td rowspan="2">样品1</td>
			<td rowspan="2">组织</td>
			<td rowspan="2">输入 DNA (ng)</td>
			<td rowspan="2">DIN2</td>
			<td rowspan="2">VAF (%)</td>
			<td rowspan="2">变体 (副本)</td>
			<td rowspan="2">参考 (复印件)</td>
			<td colspan="2">VAF (%)</td>
		</tr>
<tr>
<td>意味 着</td>
			<td>Rsd</td>
		</tr>
<tr>
<td>Ntc</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
			<td>1.23</td>
			<td>0</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
<tr>
<td>先证者</td>
			<td>血</td>
			<td>11。3</td>
			<td>7。6</td>
			<td>12。7</td>
			<td>379</td>
			<td>2.48 x 103
</td>
			<td>13。2</td>
			<td>0.0353</td>
		</tr>
<tr>
<td>父亲</td>
			<td>血</td>
			<td>10。3</td>
			<td>7。7</td>
			<td>0.0167</td>
			<td>1.08</td>
			<td>3.42 x 103
</td>
			<td>0.0341</td>
			<td>0.439</td>
		</tr>
<tr>
<td>母亲</td>
			<td>血</td>
			<td>10。8</td>
			<td>7。6</td>
			<td>0.0136</td>
			<td>0.956</td>
			<td>3.04 x 103
</td>
			<td>0.0313</td>
			<td>0.637</td>
		</tr>
<tr>
<td>Donor-1</td>
			<td>血</td>
			<td>11。7</td>
			<td>7。3</td>
			<td>-</td>
			<td>1.64</td>
			<td>3.01x 103
</td>
			<td>0.0543</td>
			<td>0.414</td>
		</tr>
<tr>
<td>Donor-2</td>
			<td>血</td>
			<td>10。5</td>
			<td>7。6</td>
			<td>-</td>
			<td>1.06</td>
			<td>2.90 x 103
</td>
			<td>0.0406</td>
			<td>0.539</td>
		</tr>
<tr>
<td>Donor-3</td>
			<td>血</td>
			<td>17。6</td>
			<td>7。9</td>
			<td>-</td>
			<td>4.24</td>
			<td>5.65 x 103
</td>
			<td>0.0713</td>
			<td>0.783</td>
		</tr>
<tr>
<td>Donor-4</td>
			<td>血</td>
			<td>12。3</td>
			<td>7。6</td>
			<td>-</td>
			<td>2.38</td>
			<td>4.16 x 103
</td>
			<td>0.0666</td>
			<td>0.557</td>
		</tr>
<tr>
<td>Donor-5</td>
			<td>血</td>
			<td>19。2</td>
			<td>7</td>
			<td>-</td>
			<td>1.48</td>
			<td>6.79 x 103
</td>
			<td>0.0213</td>
			<td>0.571</td>
		</tr>
<tr>
<td>Donor-6</td>
			<td>血</td>
			<td>14。4</td>
			<td>6。7</td>
			<td>-</td>
			<td>3.44</td>
			<td>4.67 x 103
</td>
			<td>0.0728</td>
			<td>0.729</td>
		</tr>
</tbody></table>表 4: dPCR 测定结果APC躯体嵌合体.1NTC: 无模板控制;捐献者: 健康的捐献者。2DIN: DNA 完整性数字。3Iwaizumi等。9、嗡嗡声基因组 2 15057 (2015)。4实验以三项、独立重复的3x 进行;数据表示为均值;区署: 相对标准差;不适用。表已从 Kahyo等修改。10。

Watch this Scientific Journal Video about Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format at JoVE

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

数字聚合酶链反应 (PCR) 是一个有用的工具, 高灵敏度检测单核苷酸变种和 DNA 复制数变种。在这里, 我们展示了测量人类基因组中稀有变种的关键考虑因素, 使用数字 PCR 技术与芯片内管格式。

数字聚合酶链反应法测定零星家族性腺瘤性息肉病患者的基因变异

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

digital-polymerase-chain-reaction-assay-for-genetic-variation

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

10.8K 次观看.  Hamamatsu University School of Medicine. 数字聚合酶链反应 (PCR) 是一个有用的工具, 高灵敏度检测单核苷酸变种和 DNA 复制数变种。在这里, 我们展示了测量人类基因组中稀有变种的关键考虑因素, 使用数字 PCR 技术与芯片内管格式。

视频: Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

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 ...

科研

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. ...

癌症研究

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal gastric mucosa. Recently, we detected CDH1a transcript variant in fresh-frozen tumor tissues obtained from patients with GC. The expression of this variant in tissue samples was investigated by the chip-based digital PCR (dPCR) approach presented here. dPCR offers the potential for an accurate, robust, and highly sensitive measurement of nucleic acids and is increasingly utilized for many applications in different fields. dPCR is capable of detecting rare targets; in addition, dPCR offers the possibility for absolute and precise quantification of nucleic acids without the need for calibrators and standard curves. In fact, the reaction partitioning enriches the target from the background, which improves amplification efficiency and tolerance to inhibitors. Such characteristics make dPCR an optimal tool for the detection of the CDH1a rare transcript.

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

The manuscript describes a chip-based digital PCR assay to detect a rare CDH1 transcript variant (CDH1a) in fresh-frozen normal and tumor tissues obtained from patients with gastric cancer.

基于芯片的数字 PCR 检测新鲜冷冻胃癌组织中的一个CDH1罕见转录变体

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal ...

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 ...

Evaluation of Colorectal Cancer Risk and Prevalence by Stool DNA Integrity Detection

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a reliable probability of the presence of pre-neoplastic or neoplastic colorectal lesions. This method, called fluorescence long DNA (FL-DNA), is a fast, non-invasive procedure that is an improvement upon the primary prevention system. This method is based on evaluation of fecal DNA integrity by quantitative amplification of specific targets of genomic DNA. In particular, the evaluation of DNA fragments longer than 200 bp allows for detection of patients with colorectal lesions with very high specificity. However, this system and all currently available stool DNA tests present some general issues that need to be addressed (e.g., the frequency at which tests should be carried out and optimal number of stool samples collected at each timepoint for each individual). However, the main advantage of FL-DNA is the possibility to use it in association with a test currently used in the CRC screening program, known as the immunochemical-based fecal occult blood test (iFOBT). Indeed, both tests can be performed on the same sample, reducing costs and achieving a better prediction of the eventual presence of colorectal lesions.

The presented diagnostic FL-DNA kit is a time-saving and user-friendly method to determine the reliable probability of the presence of colorectal cancer lesions.

通过大刀DNA完整性检测评估结肠直肠癌风险和患病率

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a ...

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 ...

A Robust Polymerase Chain Reaction-based Assay for Quantifying Cytosine-guanine-guanine Trinucleotide Repeats in Fragile X Mental Retardation-1 Gene

Fragile X syndrome (FXS) and associated disorders are caused by expansion of the cytosine-guanine-guanine (CGG) trinucleotide repeat in the 5&#8217; untranslated region (UTR) of the Fragile X mental retardation-1 (FMR1) gene promoter. Conventionally, capillary electrophoresis fragment analysis on a genetic analyzer is used for the sizing of the CGG repeats of FMR1, but additional Southern blot analysis is required for exact measurement when the repeat number is higher than 200. Here, we present an accurate and robust polymerase chain reaction (PCR)-based method for quantification of the CGG repeats of FMR1. The first step of this test is PCR amplification of the repeat sequences in the 5&#8217;UTR of the FMR1 promoter using a Fragile X PCR kit, followed by purification of the PCR products and fragment sizing on a microfluidic capillary electrophoresis instrument, and subsequent interpretation of the number of CGG repeats by referencing standards with known repeats using the analysis software. This PCR-based assay is reproducible and capable of identifying the full range of CGG repeats of FMR1 promoters, including those with a repeat number of more than 200 (classified as full mutation), 55 to 200 (premutation), 46 to 54 (intermediate), and 10 to 45 (normal). It is a cost-effective method that facilitates classification of the FXS and Fragile X-associated disorders with robustness and rapid reporting time.

An accurate and robust polymerase chain reaction-based assay for quantifying cytosine-guanine-guanine trinucleotide repeats in the Fragile X mental retardation-1 gene facilitates molecular diagnosis and screening of Fragile X syndrome and Fragile X-related disorders with shorter turn-around time and investment in equipment.

一种基于强健聚合酶链反应的测定法，用于定量易碎 X 智力迟钝-1 基因中的细胞氨酸-鸟-鸟-鸟- 三核苷酸重复

Fragile X syndrome (FXS) and associated disorders are caused by expansion of the cytosine-guanine-guanine (CGG) trinucleotide repeat in the 5&#8217; ...

使用 Ion Torrent 测序的 SNP 指导的胃癌 5-FU 疗法

Multi-Gene Single Nucleotide Polymorphism Detection in Gastric Cancer Based on Ion Semiconductor Sequencing Platform

Gastric cancer is a common heterogeneous tumor. Most patients have advanced gastric cancer at the time of diagnosis and often need chemotherapy. Although 5-fluorouracil (5-FU) is widely used for treatment, its therapeutic sensitivity and drug tolerance still need to be determined, which emphasizes the importance of individualized administration. Pharmacogenetics can guide the clinical implementation of individualized treatment. Single nucleotide polymorphisms (SNPs), as a genetic marker, contribute to the selection of appropriate chemotherapy regimens and dosages. Some SNPs are associated with folate metabolism, the therapeutic target of 5-FU. Methylenetetrahydrofolate reductase (MTHFR) rs1801131 and rs1801133, dihydrofolate reductase (DHFR) rs1650697 and rs442767, methionine synthase (MTR) rs1805087, gamma-glutamyl hydrolase (GGH) rs11545078 and solute carrier family 19 member 1 (SLC19A1) rs1051298 have been investigated in different kinds of cancers and antifolate antitumor drugs, which have potential forecasting and guiding significance for application of 5-FU. The ion torrent next-generation semiconductor sequencing technology can rapidly detect gastric cancer-related SNPs. Each time a base is extended in a DNA chain, an H+ will be released, causing local pH changes. The ionic sensor detects pH changes and converts chemical signals into digital signals, achieving sequencing by synthesis. This technique has low sample requirement, simple operation, low cost, and fast sequencing speed, which is beneficial for guiding individualized chemotherapy by SNPs.

作者聚焦：胃癌氟尿嘧啶反应的遗传分析

This protocol proposes holistic laboratory procedures required to detect single nucleotide polymorphisms in gastric cancer samples based on an ion semiconductor sequencing platform. The target sequences, ligating adapters, library amplification and purification, and quality control criteria are also described in detail.

基于离子半导体测序平台的胃癌多基因单核苷酸多态性检测

Gastric cancer is a common heterogeneous tumor. Most patients have advanced gastric cancer at the time of diagnosis and often need chemotherapy. Although ...

数字聚合酶链反应法测定零星家族性腺瘤性息肉病患者的基因变异

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此视频中的章节

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

基于芯片的数字 PCR 检测新鲜冷冻胃癌组织中的一个CDH1罕见转录变体

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

通过大刀DNA完整性检测评估结肠直肠癌风险和患病率

患者生物流体中肿瘤相关循环DNA的检测与监测

一种基于强健聚合酶链反应的测定法，用于定量易碎 X 智力迟钝-1 基因中的细胞氨酸-鸟-鸟-鸟- 三核苷酸重复

基于离子半导体测序平台的胃癌多基因单核苷酸多态性检测

基于离子半导体测序平台的胃癌多基因单核苷酸多态性检测

基于离子半导体测序平台的胃癌多基因单核苷酸多态性检测