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
Summary
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。
Abstract
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.
Introduction
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 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的DNA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“>移液到96孔板中，数据分析是用软件来执行时。
Protocol
Representative Results
Discussion
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…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Hamilton MICROLAB STARlet IVD instrument | Siemens | 10701001 | Automated DNA isolation instrument |
| QX200 Droplet Generator | Bio-Rad | 772BR1119 | |
| QX200 Droplet Reader | Bio-Rad | 771BR1497 | |
| Conventional PCR machine capable of ramp-time adjustment | 621BR17718 | ||
| PX1 PCR plate sealer | Bio-Rad | 770BR1575 | |
| QuantaSoft software | Bio-Rad | ||
| DNA isolation kit | |||
| VERSANT Tissue Preparation Reagents Box 1 | Siemens | 10632398 | |
| VERSANT Tissue Preparation Reagents Box 1 | Siemens | 10632399 | |
| CO-RE tips | Siemens | ||
| ddPCR mutation analysis | |||
| ddPCR Supermix | Bio-Rad | BR186-3010 | 2X concentration |
| DG8 cartridge | Bio-Rad | BR186-4008 | |
| Droplet Generator oil | Bio-Rad | BR-186-3005 | |
| Gasket | Bio-Rad | BR186-3006 | |
| Droplet reader oil | Bio-Rad | BR-186-3004 |
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