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DNA测序技术的发展：第三代测序技术

发布: 2015-11-04 1,128阅读 209评论

本文作为DNA测序的最终章，就和大家聊一聊最新一代的DNA测序技术。不相同于上两篇文章中提到的桑格法测序和SBS高通量测序，小编并没有给第三代测序法一个简单的概括性命名。原因是第三代测序的发展方向太多，不好直接概括为某一特定方法。具体来说，在第二代测序的基础上，人们还希望提高测序效率、提高测序通量、提高测序准确率、避免PCR扩增和避免荧光检测等等。比如，Pacific BioSciences公司的实时单分子测序（real-time single-molecule）和complete genomics公司的复合探针-锚定连接技术（combinatorial probe-anchor ligation，cPAL），都依靠荧光检测来测序，但他们提高的是测序速度和通量；Life technologies公司的离子流探测测序设备（Ion torrent）使用离子流场效应半导体感应器（ion-sensitive field effect transistor）,依靠边合成边测序的中心概念，检测每次添加碱基时候释放出的离子流，从而避免了传统第二代的荧光检测；Oxford nanopore公司的纳米孔单分子测序技术，同样也避免了荧光检测和对主体DNA序列的PCR扩增。

 

第三代测序技术

 

PacBio 实时单分子测序

中心思想也是边合成边测序，四种分别荧光标记的dNTPs参与到DNA聚合酶主导的链合成反应中，每类碱基在被添加上去的时候，会显示不同的荧光。当把这个链合成反应控制在一个DNA母板链、一个DNA聚合酶，一个相对封闭的反应空间的时候，就可以方便地对每次加入的荧光进行判别。PacBio公司采用零模波导纳米孔（zero-mode waveguide nanostructure）来盛装单一的链合成反应，如图1所示。每次被激发的散射光会被收集并分析波长，以判断荧光颜色，进而推断出碱基类型。

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图1： PacBio 实时单分子测序技术模拟图【1】

 

Complete Genomics 公司的复合探针-锚定连接技术

这项技术有点像升级版的SOLiD系统，但比之更复杂，这次是一次性结合9个碱基，只有第五个碱基是确定的A、T、C或G，而其他8个碱基则是随机的。所以每个探针的长度是9个碱基，荧光种类还是4种，有每个探针的第五个碱基的种类确定。具体过程如图2。

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图2：（A）整个反应的流程，从制备DNA库开始，到形成环状DNA样板，再到构建纳米级的锚定位列，最后测序分析。（B）一段长400bp的DNA片段被添加了四种特定序列，整个区域被分成八块，这样的细分可以方便后来在硅板上的特定结合和细化的测序。（C）纳米位列整合，每个点都有结合一个与其他纳米点不一样的短链DNA锚，可以方便环状DNA片段的特异性结合。（D）探针与锚定的环状DNA特异性结合，采集荧光，分析序列，即cPAL技术。【2】

 

Oxford Nanopore 纳米孔单分子通道技术

纳米孔技术的基本原理是探测电信号而不是荧光信号。这个技术的核心是设计出了可以允许单个碱基通过的蛋白纳米通道，每种碱基通过通道的时候会对通道内的电流和通道两侧的电压产生不同的微小影响。采用灵敏的电子设备可以检测到这些变化，进而判断出通过纳米通道的碱基类型（如图3所示）。Nanopore的理念是Lab-on-Chip，所以他们一直致力于单分子的测序的研发，也算是第三代测序技术中突破传统，打开新理念的一种。

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图3：纳米孔单分子通道【3】

 

Ion Torrent 电子流检测技术

这也是新一代测序技术中并不用到荧光检测的一种，它检测的是在合成过程中，不同碱基加入而释放出的不同强弱的离子流，如图4所示。

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图4：电子流检测【1】

总之，新一代的测测序技术都朝着单分子测序在发展，未来的目标是让测序反应小规模化，精准化而且高效化。小编认为未来的前景还是十分广阔的，当基因组测序成本降到1000美元以下的时候，我们才真正迎来了解密基因的时代啊。

 

References:

【1】Niedringhaus, T. P., Milanova, D., Kerby, M. B., Snyder, M. P., & Barron, A. E. (2011). Landscape of next-generation sequencing technologies. Analytical chemistry, 83(12), 4327-4341.

【2】Drmanac, R., Sparks, A. B., Callow, M. J., Halpern, A. L., Burns, N. L., Kermani, B. G. & Sharanhovich, U. (2010). Human genome sequencing using unchained base reads on self-assembling DNA nanoarrays. Science, 327 (5961), 78-81.

【3】Clarke, J., Wu, H. C., Jayasinghe, L., Patel, A., Reid, S., & Bayley, H. (2009). Continuous base identification for single-molecule nanopore DNA sequencing. Nature nanotechnology, 4(4), 265-270.

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