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
Summary
在这里, 我们提出了一个协议, 以准确地量化一个目标区域的多基因变化, 在单一的反应使用下拉 ddPCR 和一个独特的对水解探针。
Abstract
液滴数字聚合酶链反应 (ddPCR) 是一种高度敏感的定量聚合酶链反应 (PCR) 方法, 以样品分馏为基础, 以数以千计的纳米水中油的个体反应。最近, ddPCR 已成为循环肿瘤 DNA (ctDNA) 检测最准确、最灵敏的工具之一。标准 ddPCR 技术的主要限制之一是限制数量的突变, 可以筛选每个反应, 因为特定的水解探针承认每个可能的等位基因版本是必需的。另一种方法, 即下拉 ddPCR, 增加了吞吐量, 因为它只需要一双探针来检测和量化目标区域中可能发生的所有基因改变。ddPCR 对常规 ddPCR 的检测具有相当的敏感性, 其优点是在单个反应中发现更多的突变。它是成本效益, 保存宝贵的样品材料, 也可以作为一个发现工具, 当突变是未知的先验。
Introduction
数以千计的躯体突变与癌症的发展已经报告了1。其中, 有几个是靶向治疗2、3的疗效预测指标, 这些突变的基因筛选现在是常规的临床实践。液滴数字 PCR (ddPCR) 技术可用于监测存在或没有突变的高检测精度, 并高度兼容非侵入性液体活检4,5。然而, 目前的 ddPCR 化验主要是为了检测已知的突变先验。当突变未知时, 这严重限制了 ddPCR 作为发现工具的使用。事实上, 在传统的 ddPCR 设计中, 识别野类等位基因的水解探针与识别突变等位基因 (物探针) 的特定探针竞争 (图 1A)。具有较高亲和性的探针 hybridizes 模板并释放其荧光, 表明相应的等位基因的性质。为每个液滴获得的荧光数据可以在一个散射图中可视化, 表示由物探针在不同尺寸中发射的荧光。在图 1A中, 给出了常规 ddPCR 检测的典型结果的示意图表示法。在本例中, 蓝云对应于含有由小波探针识别的物等位基因的水滴, 而红云对应于由物探针放大和识别的水滴。根据反应中载入的 DNA 数量, 物 amplicons 的双正液滴会出现 (绿云)。浅灰色云对应于空滴。
由于大多数平台允许检测有限数量的显影 (通常为 2, 但高达 5), 常规 ddPCR 的吞吐量是有限的。因此, 针对有多个相邻突变的区域, 需要设计技术上具有挑战性的多重 ddPCR 检测, 或使用针对每种突变的多个反应并行。ddPCR 策略, 克服了这一限制, 因为它可以在一个目标区域内使用一个独特的对小波水解探针来检测任何基因改变。第一探针 (探头 1) 覆盖一个非变量序列, 与目标区域相邻, 第二探针 (探头 2) 与预期的突变目标区域的小波序列互补 (图 1B)。当第一探针量化反应中的 amplifiable 分子总量时, 第二探针通过亚优杂交对突变序列进行物等位基因的鉴别。探针2可以识别目标区域中的多种突变 (单个或多个核苷酸替代、删除等)6。与传统的 ddPCR 一样, 浅灰色云对应于不含 DNA 分子的水滴。重要的是要记得, 在这种类型的检测, 最佳分离的重量和物云是取决于数量的 DNA 负载在反应中, 因为含有重量和物目标等位基因的水滴不能区别于只含有重量的水滴形式。因此, 这种化验要求大多数的水滴包含的目标基因不超过一份。
Protocol
Representative Results
Discussion
为了设计一种高效的降 ddPCR 检测方法, 优化是至关重要的, 必须严格遵循该协议。引物和探针的每个组合都具有独特的 PCR 反应效率。因此, 在对有价值的测试样品使用之前, 必须对对照样品进行仔细的验证。优化和验证是保证峰值信号检测和评估特异性和灵敏度的重要因素。如《议定书》所述, “探测 2” 所涵盖的目标区域内的所有突变都可能被审问。尽管如此, 建议对位于 “探针 2” 的 5 ‘ 或 3 ‘ 端点?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了居里 SiRIC (格兰特 DGOS-4654) 的支持。作者还希望感谢卡罗琳 Hego 对视频的贡献。
Materials
| K2EDTA tube (color code : lavender) | BD | 367863 | EDTA tubes are used to obtain a whole blood or EDTA plasma sample |
| QIAamp Circulating Nucleic Acid Kit (manual protocol) | Qiagen | 55114 | For isolation of free-circulating DNA from human plasma |
| QIAsymphony DSP Circulating DNA Kit (automated protocol) | Qiagen | 937556 | For isolation of free-circulating DNA from human plasma |
| QIAsymphony SP system | Qiagen | 9001297 | fully integrated and automated system for cfDNA, DNA or RNA purification |
| QIAvac 24 Plus | Qiagen | 19413 | For purification of up to 24 cfDNAs simultaneously |
| Qubit fluorometer | Invitrogen | Q33226 | The Qubit fluorometer is a benchtop fluorometer for the quantitation of DNA |
| QX100 or QX200 reader | Bio-Rad | 186-3003 or186-4003, respectively | The reader measures fluorescence intensity of each droplet and detects the size and shape as droplets pass the detector |
| QX100 or QX200 droplet generator | Bio-Rad | 186-3002 or 186-4002, respectively | Instrument used for droplet generation |
| C1000 thermal cycler | Bio-Rad | 1851196 | Modular thermal cycler platform, includes C1000 Touch thermal cycler chassis, 96-well fast reaction module |
| Plate sealer | Bio-Rad | 181-4000 | PX1™ PCR plate sealer |
| DG8 cartridge holder | Bio-Rad | 186-3051 | Positions and holds the DG8 cartridge in the instrument for droplet generation |
| Droplet generator cartridges and gaskets | Bio-Rad | 186-4007 | Microfluidic DG8 cartridge used to mix sample and oil to generate droplets; DG8 gaskets seal the cartridge to prevent evaporation and apply the pressure required for droplet formation |
| PCR supermix | Bio-Rad | 186-3010 | ddPCR supermix for probes (no dUTP) |
| 96-well PCR plates | Eppendorf | 951020362 | 96-well semi-skirted plates |
| Foil seal | Bio-Rad | 181-4040 | Pierceable foil heat seal |
| ddPCR Droplet Reader Oil | Bio-Rad | 186-3004 | oil used in the read |
| Droplet Generation Oil for Probes | Bio-Rad | 186-3005 | oil for droplet generation |
| DNA loBind Tube 1.5 mL | Eppendorf | 0030 108.051 | Eppendorf LoBind Tubes maximize sample recovery by significantly reducing sample-to-surface binding |
| Multiplex I cfDNA Reference Standard Set | HORIZON | HD780 | The Multiplex I cfDNA Reference Standards are highly-characterized, biologically-relevant reference materials used to assess the performance of cfDNA assays that detect somatic mutations |
| QUBIT DSDNA HS ASSAY KIT, 500 | Life Technologies | Q32854 | The HS assay is highly selective for double-stranded DNA (dsDNA) over RNA and is designed to be accurate for initial sample concentrations from 10 pg/µL to 100 ng/µL |
| Qubit Assay Tubes | Life Technologies | Q32856 | Qubit assay tubes are 500 µL thin-walled polypropylene tubes for use with the Qubit Fluorometer |
Riferimenti
- Alexandrov, L. B., Nik-Zainal, S., et al. Signatures of mutational processes in human cancer. Nature. 500 (7463), 415-421 (2013).
- Amado, R. G., Wolf, M., et al. Wild-Type KRAS Is Required for Panitumumab Efficacy in Patients With Metastatic Colorectal Cancer. Journal of Clinical Oncology. 26 (10), 1626-1634 (2008).
- Karapetis, C. S., Khambata-Ford, S., et al. K-ras mutations and benefit from cetuximab in advanced colorectal cancer. New England Journal of Medicine. 359 (17), 1757-1765 (2008).
- Postel, M., Roosen, A., Laurent-Puig, P., Taly, V., Wang-Renault, S. -. F. Droplet-based digital PCR and next generation sequencing for monitoring circulating tumor DNA: a cancer diagnostic perspective. Expert Review of Molecular Diagnostics. 18 (1), 7-17 (2018).
- Saliou, A., Bidard, F. -. C., et al. Circulating tumor DNA for triple-negative breast cancer diagnosis and treatment decisions. Expert Review of Molecular Diagnostics. 16 (1), 39-50 (2015).
- Decraene, C., Silveira, A. B., et al. Multiple Hotspot Mutations Scanning by Single Droplet Digital PCR. Clinical chemistry. 64 (2), 317-328 (2018).
- Untergasser, A., Cutcutache, I., et al. Primer3–new capabilities and interfaces. Nucleic Acids Research. 40 (15), e115 (2012).
- Snyder, M. W., Kircher, M., Hill, A. J., Daza, R. M., Shendure, J. Cell-free DNA Comprises an In Vivo Nucleosome Footprint that Informs Its Tissues-Of-Origin. Cell. 164 (1-2), 57-68 (2016).
- Bidshahri, R., Attali, D., et al. Quantitative Detection and Resolution of BRAF V600 Status in Colorectal Cancer Using Droplet Digital PCR and a Novel Wild-Type Negative Assay. The Journal of Molecular Diagnostics. 18 (2), 190-204 (2016).
- Attali, D., Bidshahri, R., Haynes, C., Bryan, J. ddpcr: an R package and web application for analysis of droplet digital PCR data. F1000Research. 5, (2016).
- Forbes, S. A., Beare, D., et al. COSMIC: somatic cancer genetics at high-resolution. Nucleic Acids Research. 45 (D1), D777-D783 (2017).
