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DNA测序技术的发展：第二代SBS技术

发布: 2015-11-04 404阅读 117评论

在上一篇文章中，小编提到的初代桑格测序法，兴起于上世纪七十年代，发展到现在已经不能算是主流的测序技术了，但桑格法激励和启发了一代科学家们去研发高通量、高准确度的新一代测序技术，这才有了现在的第二代和第三代测序技术。相比于桑格测序法，第二代、第三代的进步是更快、更便宜而且保持了准确率。举个例子，以前使用桑格法测序人类基因组需要三年的时间，而现在运用第二代测序技术，仅仅需要一周时间。在本文，小编将和大家聊聊第二代SBS技术(SBS：sequencing-by-synthesis，边合成边测序），而在接下来的测序技术系列文章终结篇中，我们再来看看第三代测序技术和DNA测序的未来。

第二代测序技术

初代桑格法测序读长可一次性达到1000 bp，而且准确率高达99.999%，但是桑格法牵扯到跑琼脂糖胶读碱基，导致其成本过高，而且通量很低，所以桑格法并不适用于大规模商业应用。经过多方面的改进和开发，第二代测序技术已经非常成熟，而已经大规模用于商业测序。以Illumina 公司的Solexa和Hiseq系统、Roche公司的454系统以及ABI公司的SOLiD系统为代表，第二代测序技术确实大大降低了测序成本，缩短了测序时长。

Illumina Solexa 系统

Illumina公司的Solexa和Hiseq系统的基本原理是一致的，都是采用边合成边测序的方法，是目前市面上使用量最大的测序系统。测序过程可以简单概括为四个阶段，详细过程如下图所示：

YL01

1. 构建短链片段DNA库 （图中第1、2步）

利用超声波把待测的DNA样本打断成小片段，主要是打断成200-500bp长的片段，并在这些小片段的两端添加上不同的接头引物（Adapter），构建出单链DNA文库。

2. DNA片段吸附 （图中第3步）

当短链DNA通过flowcell的时候会随机附着在flowcell表面的槽道中。每个Flowcell有8个槽道，每个槽道的表面都附有很多接头，这些接头能和建库过程中加在DNA片段两端的接头相互配对（这就是为什么flowcell能吸附建库后的DNA的原因），并能支持DNA在其表面进行后续的桥式PCR的扩增（Bridge-PCR）。

3. 桥式PCR扩增 （图中第4步）

桥式PCR以Flowcell表面所固定的接头为引物，桥接后扩增（如上图第4步）。经过不断的扩增和变性循环，最终每个DNA片段都将在各自的位置上集成一束，每一个束都含有单个DNA模板的很多分拷贝，进行这一过程的目的在于实现将碱基的信号强度放大，以达到测序所需的信号要求。

4. 荧光测序 （图中第5、6步）

向反应体系中同时添加DNA聚合酶、特定一端的接头引物和不同荧光标记的4种dNTP（如同Sanger测序法）。这些dNTP的3’-OH被保护，因而每次只能添加一个dNTP，DNA链复制反应就会停止。在dNTP被添加到合成链上后，所有未使用的游离dNTP和DNA聚合酶会被洗脱掉。接着，再加入激发荧光所需的缓冲液，用以激光激发荧光信号，搜集信号记录在计算机中并找出对应碱基。这样荧光信号记录完成后，再加入化学试剂淬灭荧光信号并去除dNTP 3’-OH保护基团，以便能进行下一轮的测序反应。这种测序技术每次只添加一个dNTP的特点能够很好的地解决同聚物长度的准确测量问题，它的主要测序错误来源是碱基的替换，目前它的测序错误率在1%-1.5%之间。

Roche 454 系统

Roche 454也是第二代测序系统中比较成功的一个，基本原理也是边合成边测序。其DNA库制备方法和测序方法与Solexa有很大不同，但中心原理是相似的。基本可以分成三个阶段，如下图所示：

YL02

1. 构建短链片段DNA库 （上图第1、2步）

和illumina的不同，454系统是利用喷雾法将待测DNA打断成300-800bp长的小片段，并在片段两端加上不同的接头，或将待测DNA变性后用杂交引物进行PCR扩增，连接载体，构建单链DNA库。

2. 乳液PCR扩增（Emulsion PCR）（上图第3、4步）

454所用的DNA扩增方法也和illumina的截然不同，它将这些单链DNA结合在水油包被的直径约28um的磁珠上，并在磁珠上进行PCR扩增。乳液PCR最大的特点是可以形成数目庞大的独立反应空间。这里的关键技术是注“水”到“油”，基本过程是在PCR反应前，将包含PCR所有反应成分的水溶液注入到高速旋转的矿物油表面，水溶液瞬间形成无数个被矿物油包裹的小水滴。这些小水滴就构成了独立的PCR反应空间。在理想状态下，每个小水滴只含一个DNA模板和一个磁珠，而这个水珠是被油包裹的，整个体系里有无数个这样的小水珠。每个小水珠就是一个独立的PCR反应体系，经过扩增，每个小片段都将被扩增约100万倍，单个磁珠上的所有片段理论上应该都是一样的，从而达到下一步测序所要求的DNA量。

3. 荧光测序 （上图第5、6步）

每个磁珠上的DNA片段被DNA聚合酶预先处理，然后将磁珠放在一种PTP （Pico Titer Plate）平板上。PTP板上有许多直径约为44um的小孔，每个小孔仅能容纳一个磁珠，通过这种方法来固定每个磁珠的位置，以便收集信号。

4. 焦磷酸测序法

每次反应加入一种dNTP进行DNA链合成反应，如果dNTP能与待测序列配对，则会在合成后释放焦磷酸基团。释放的焦磷酸基团会与反应体系中的ATP硫酸化学酶（ATP sylfurylase）反应生成ATP。生成的ATP和荧光素酶（luciferase）共同氧化使测序反应中的荧光素分子（luciferin）并发出荧光。由于每一种dNTP在反应中产生的荧光颜色不同，因此可以根据荧光的颜色来判断被测分子的序列。反应结束后，游离的dNTP会在双磷酸酶的作用下降解ATP，从而导致荧光淬灭，以便使测序反应进入下一个循环。由于在PTP板上，每个测序反应都在独立的小孔中进行，因而能大大降低相互间的干扰和测序偏差。454系统最大的优势在于其能获得较长的测序读长，当前454技术的平均读长可达400bp。与illumina的Solexa和Hiseq系统相比，它最主要的一个缺点是无法准确测量同聚物的长度，如果序列中存在类似于Poly-A的情况时，测序反应会一次加入多个T，而所加入的T的个数只能通过荧光强度来推测，这就有可能导致结果不准确。

ABI SOLiD 系统

与Solexa和454都不相同，SOLiD系统利用的并不是DNA聚合酶，而是DNA链接酶。更不一样的是，SOLiD系统是每次测两个碱基，并不是一个，相当于每个目标碱基被测了两次，SOLiD是目前第二代测序中最为准确的一个。有兴趣的童鞋可以去看看SOLiD的基本原理，比上面的两种系统都要神奇。

图片来网络，文字部分由BioEngX原创。转载请注明。

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