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

Tomoaki Kahyo; Haruhiko Sugimura

doi:10.3791/58199

JoVE Journal
Cancer Research

This content is Free Access.

JoVE Journal Cancer Research

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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05:58 min

August 20, 2018

DOI: 10.3791/58199-v

Tomoaki Kahyo¹, Haruhiko Sugimura¹

¹Department of Tumor Pathology,Hamamatsu University School of Medicine

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Overview

This article discusses the application of digital polymerase chain reaction (PCR) for the sensitive detection of single nucleotide variants and DNA copy number variants. It highlights the methodology and implications of using digital PCR in genetic testing, particularly for tumor diagnosis.

Key Study Components

Area of Science

Genetics
Oncology
Diagnostic Techniques

Background

Digital PCR allows for high-throughput detection of low-variant allele fractions.
This technique is beneficial for detecting mosaicism and in liquid biopsy-based genetic testing.
It contributes to precision medicine by enabling the detection of oncogenic mutations with high sensitivity.

Purpose of Study

To demonstrate the effectiveness of digital PCR in measuring rare genetic variants.
To provide a detailed protocol for implementing this technique.
To explore the implications of digital PCR in clinical diagnostics.

Methods Used

Preparation of DNA samples and mixing with primers and probes.
Electrophoresis for initial DNA analysis.
Utilization of a thermal cycler for PCR amplification.
Detection of PCR products using fluorescence intensity analysis.

Main Results

Successful detection of APC allele variants in patient samples.
Variant allele fraction (VAF) calculated by digital PCR was comparable to next-generation sequencing results.
Electropheresis confirmed the size of PCR products.
Demonstrated the importance of maintaining a clean workspace during the procedure.

Conclusions

Digital PCR is a powerful tool for detecting rare genetic variants.
This method has significant implications for cancer diagnosis and precision medicine.
Proper execution of the protocol is crucial for accurate results.

Frequently Asked Questions

What is digital PCR?

Digital PCR is a technique used to amplify and quantify DNA, allowing for the detection of rare genetic variants with high sensitivity.

How does digital PCR differ from traditional PCR?

Unlike traditional PCR, digital PCR partitions the sample into thousands of individual reactions, enabling precise quantification of low-abundance targets.

What are the applications of digital PCR?

Digital PCR is used in genetic testing, cancer diagnostics, and research involving rare genetic variants.

What is the significance of variant allele fraction (VAF)?

VAF indicates the proportion of a specific variant in a sample, which is crucial for understanding the genetic landscape of tumors.

What precautions should be taken during digital PCR?

Maintaining a clean workspace and following the protocol meticulously are essential to avoid contamination and ensure accurate results.

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。

这种方法可以帮助回答基因检测领域的关键问题，例如嵌合体检测和基于液体活检的基因检测。该技术的主要优点是低变异等位基因分数的高通量检测。这项技术的影响延伸到肿瘤的诊断，因为以高灵敏度检测致癌突变有助于精准医疗。

通过将 10 L DNA 样品缓冲液添加到 8 管联管的试管中开始此实验。然后向 8 管联管的第一管中加入 1 μL DNA 分子量标准，向其余每管中加入 1 μL 样品基因组 DNA。使用微孔板附件通过涡旋 1 分钟混合 8 管联排的内容物。

将试纸条、移液吸头和凝胶装置放入电泳仪中。按下 start 按钮开始运行。运行完成后，检查显示屏上的电泳图，以确认 DNA 样品缓冲液中包含的下部标记物在电泳图上分配正确。

如果没有，请在软件的电泳图模式下手动分配标记。然后，在软件的区域模式下，指定基因组 DNA 的大小区域以自动计算 DNA 的浓度。将先前设计的引物、探针和基因组 DNA 添加到新的 8 管联管中，以达到 15 L.Pipette 上下混合的总体积。

将加载平台添加到新 8 联管中内置的芯片上，然后将联管放入自动进样器中。确保芯片和装载平台之间有接触。接下来，在平台上放置一个加载滑块，并使用塞子将滑块从加载器上固定下来。

在

滑块尖端附近移取 15 L PCR 混合物，然后按下上样器按钮运行上样器一分钟。运行后从加载器中取出试管条，并将其放入密封增强剂中。小心地推动滑盖和顶盖的边缘。

运行密封增强剂约 2 分钟。如果密封不完全，如一滩液体所示，请再重复运行一分钟。向试管中加入 230 L 密封液。

将试管联排管放入热循环仪中，然后按照实验步骤中的说明运行 PCR。如果阳性分区分布不均匀，请调整温度或 PCR 的持续时间。为了检测和分析 PCR 产物的荧光强度，将试管条放在检测夹具上，并加入 6 mL 蒸馏水。

使用移液器吸头去除任何可见的气泡。将夹具装入检测器。在检测软件中，选择 Fluorescence（荧光）、Experiment（实验），然后选择 Sample NTC（样品 NTC）选项卡，然后单击 RUN（运行）按钮开始运行。

运行完成后，确认位置图、直方图和 2-D 散点图。要收集 PCR 产物，请从试管中取出密封液。加入 100 L TE 缓冲液，剧烈涡旋 30 秒。

在台式离心机中短暂离心试管，然后按照文本方案中的说明进行，以完成 PCR 产物的收集。通过数字 PCR 创建的位置图在患者和父亲样本中均显示黄色 HEX 荧光，表明存在 APC 等位基因变体 T，但在无模板对照中不存在。只有患者的基因组 DNA 含有 C 变体，如 FAM 的绿色荧光所示。

散点图进一步证实，患者和父亲的 HEX 或变异 T 均呈阳性，而只有患者显示存在变异 C.通过 DPCR 计算的患者变异等位基因分数或 VAF 为 13.2%，与下一代测序实现的 12.7% 相似。另一方面，患者父母和健康供体的 VAF 小于 0.1%收集和浓缩的 DPCR 产物的电泳证实，预测片段的大小在患者和父母的 DNA 中为 123 个碱基对。在尝试此过程时，请务必记住保持工作台清洁，例如使用风扇过滤器装置。