PNAS、Nature共造基因测序新方法,不“放过”任何碱基
2016/04/25
日前,来自哥伦比亚大学、哈弗大学及美国国家标准技术局的研究人员报道了使用蛋白纳米孔阵列实现了单碱基分辨率的实时单分子电子DNA测序,相关结果发表在PNAS杂志上。


日前,来自哥伦比亚大学、哈弗大学及美国国家标准技术局的研究人员报道了使用蛋白纳米孔阵列实现了单碱基分辨率的实时单分子电子DNA测序,相关结果以《Real-Time Single Molecule Electronic DNA Sequencing by Synthesis Using Polymer Tagged Nucleotides on a Nanopore Array》为题发表在PNAS杂志上。

基因测序是实现个性化医疗和精准医学的关键技术,推动了生物医学领域的快速发展,完整的个人基因组序列为医疗诊断及医疗保健提供了重要的标志和指导方针。目前,获取高精度序列面临着成本和速度两大挑战。在过去的十几年中,测序取得了很大的进展,目前广泛使用的高通量技术依赖于对DNA四个碱基的光学检测,为了探讨可替代的检测技术,逐渐发展起DNA模板的电子测序,并应用于遗传学分析。

PNAS和Nature子刊:实时单分子电子DNA测序

近两年开发的纳米孔测序,依赖于单链DNA通过纳米孔时在电压的作用下产生了可用于序列检测的电子信号,然而,由于DNA四个碱基的化学结构非常相似,因此不容易通过这种方法来区分,因此研究者们一直致力于寻找和发展一个精准的单分子电子DNA测序平台,以生产一种小型化的能够破译基因组的DNA测序仪。

2012年,研究人员曾在《Scientific Reports》报道了纳米孔测序单分子边合成边测序(SBS)的原理。如今,研究人员在PNAS上描述了利用完整的SBS系统产生的单碱基水平单分子电子测序数据。SBS系统通过在四个碱基分别标记上不同的聚合物,聚合酶会将标记的核苷酸添加到DNA链上,同时释放出聚合物标签,这些标签通过纳米孔时引起电流变化,根据不同标签造成的电信号差异,可以准确分辨出相应的碱基。

研究人员将聚合酶栓系在α-hemolysin纳米孔上,在芯片上排除纳米孔阵列,随后用多重化的纳米孔传感器搭建了高通量的测序平台,该平台使用多聚物标记的核苷酸,可在单分子水平上对多个DNA模板进行平行测序,通过这种方式,研究人员获得了实时的单分子测序数据。

优化措施多,发展空间大

研究人员表示,“纳米孔SBS方法的新颖性要从设计、合成及四种碱基不同聚合物标记的选择讲起,我们用DNA聚合酶共价连接到纳米孔和聚合物标记物的碱基上,在DNA复制过程中,带有聚合物标记的碱基通过碱基互补原则连接到待测序的链上,通过纳米孔时释放出独特的信号,就是这种独特信号帮助我们实现实时单分子测序,同时克服了纳米孔测序面临的障碍。此外,可对聚合物标记的大小、电荷和结构进行改进,从而实现最佳分辨率的SBS系统,该项目为精准医学提供了前所未有的具有成本效益的遗传诊断平台”,

据研究人员表示,目前基于SBS的测序仪所产生的数据远远超过PNAS所报道。最近已经实现了超过1000个碱基的读取长度。谈及未来,研究人员表示将继续调整连接器及在分子水平上调整聚合物标记的结构和电荷来优化该系统,希望最终能在该系统上实现准确、快速和低成本的全人类基因组测序,并用于常规的医学诊断。

相关阅读:

Team advances single molecule electronic DNA sequencing

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  • PEG-Labeled Nucleotides and Nanopore Detection for Single Molecule DNA Sequencing by Synthesis

    We describe a novel single molecule nanopore-based sequencing by synthesis (Nano-SBS) strategy that can accurately distinguish four bases by detecting 4 different sized tags released from 5?-phosphate-modified nucleotides. The basic principle is as follows. As each nucleotide is incorporated into the growing DNA strand during the polymerase reaction, its tag is released and enters a nanopore in release order. This produces a unique ionic current blockade signature due to the tag's distinct chemical structure, thereby determining DNA sequence electronically at single molecule level with single base resolution. As proof of principle, we attached four different length PEG-coumarin tags to the terminal phosphate of 2?-deoxyguanosine-5?-tetraphosphate. We demonstrate efficient, accurate incorporation of the nucleotide analogs during the polymerase reaction, and excellent discrimination among the four tags based on nanopore ionic currents. This approach coupled with polymerase attached to the nanopores in an array format should yield a single-molecule electronic Nano-SBS platform.

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  • Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array

    DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a single-molecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5′-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotide-based polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods.

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