收集和唾液DNA提取用于新一代测序
Summary
DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.
Abstract
The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.
Introduction
获得高质量的DNA对人类遗传研究是在疾病基因的发现进程至关重要。血，虽然需要侵入性手术，也比唾液采集更昂贵，是有利的，用于创建永生化细胞系的DNA的无限源，或iPS细胞的功能性研究，有时血液的DNA时使用的细胞系是不可用的。然而，获得的血液需要经过培训的采血和血液有一个较短的半衰期比口水1。从唾液DNA是较便宜，容易得到，因为它可以被收集，并通过邮件发送，而不需要用于抽血，由此增加潜在受试者池远远超出医院和实验室2的集水面积。当受试者具有给人一种唾液样品代替血液3,4的选择研究登记可以得到改进。左右的数量，从唾液DNA的质量问题可能已限制了其广泛我们ë尽管许多研究最近的研究显示全唾液的适用性，平均每毫升4.3×10 5个细胞，用于对较早的口腔拭子的方法未获得显著量唾液2，3，4，5的DNA检测， 6，虽然适度的文学存在呈现出唾液的DNA是否适合进行基因分型应用，包括基于微阵列的方法，8，9，10，没有研究探讨了新一代测序（NGS）。为优化这种唾液DNA提取协议的目的是最大限度地提高数量和质量的遗传学应用，很容易与普通试剂和耗材实验室实现了一个具有成本效益的方式。
从唾液DNA提取需要几个步骤：1）收集并存储，2）细胞裂解，3）核糖核酸酶处理，4）沉淀蛋白，5）用乙醇沉淀，6）DNA的补液。该DNA的稳定缓冲液，所述的此前2，没有充分改变功能。没有尝试优化核糖核酸酶处理和DNA补液措施作出。对于每一个剩下的步骤，可能影响产量的几个变量进行鉴定。每个变量都被单独操纵和提高产量和质量统计评估。对于被证实可以改善产率和/或DNA质量的变量的最优值被包括在最终的协议。
Protocol
Representative Results
Discussion
本程序是一个优化的DNA提取的协议，已大大改善了高分子量DNA的产量相比，标准的方法，而不损害DNA的质量。与对产量的大部分影响最大的关键步骤是5.2步，其中包括在乙醇沉淀比任何出版协议审查这里，除了一个没有广泛分布11较长的离心步骤。检测DNA的质量没有改变与该较长的离心关联，表明大部分从唾液采集的可用的DNA是不是退化和高的分子量。
唾液收集在样?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was funded by a National Institutes of Health R01 (DC009453 support to CWB).
Materials
| 15ml Centrifuge Tubes | Fisher | 12-565-268 |
| Cell Lysis Solution | Qiagen | 158908 |
| Proteinase K | Sigma | P6556 |
| Protein Precipitation Solution | Qiagen | 158912 |
| Isopropanol | Fisher | A416-4 |
| Glycogen | EZ-BioResearch | S1003 |
| 70% Ethanol | Fisher | 04-355-305 |
| Tris-EDTA (TE) | Fisher | BP2473-1 |
| NaCl | Fisher | AC194090010 |
| Tris HCl | Fisher | BP1757-100 |
| EDTA(0.5M) Solution | Fisher | 03-500-506 |
| Sodium Dodecyl Sulfate | Fisher | BP166-100 |
| Equipment | ||
| Name of Equipment | Distributor | Catalog# |
| Analog Vortex Mixer | Fisher | 02-215-365 |
| Centrifuge 5810R | Eppendorf | 5811 000.010 |
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