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Nature:纳米孔测序技术的发明人再发明单分子互作测序

2014-10-04 MedSci MedSci原创

随着技术的发现,大规模并行DNA测序得到了广泛的应用,为许多研究领域带来了一场革命。然而,高通量的蛋白质分析仍然困难重重,现在亟需高质量低成本的蛋白分析技术。为此,遗传学界的大牛George M. Church领导哈佛医学院的团队,开发了一种单分子互作测序(SMI-seq)技术。该技术能够实现单分子水平上的并行分析,获得大量蛋白质的互作图谱。这一成果发表在近期的Nature杂志上,文章的通讯作者是

随着技术的发现,大规模并行DNA测序得到了广泛的应用,为许多研究领域带来了一场革命。然而,高通量的蛋白质分析仍然困难重重,现在亟需高质量低成本的蛋白分析技术。

为此,遗传学界的大牛George M. Church领导哈佛医学院的团队,开发了一种单分子互作测序(SMI-seq)技术。该技术能够实现单分子水平上的并行分析,获得大量蛋白质的互作图谱。这一成果发表在近期的Nature杂志上,文章的通讯作者是哈佛医学院的George M. Church和Liangcai Gu。

研究人员利用PRMC复合体(蛋白质-核糖体-mRNA-互补DNA),通过体外的核糖体展示(ribosome display)技术,将DNA条码连到蛋白质上。此外也可以通过催化酶,分别给不同蛋白连上DNA条码。这些自带条码的蛋白可以在水溶液中进行检测。

随后,研究人员将上述蛋白固定在聚丙烯酰胺薄膜上,建立起随机的单分子阵列。对条码DNA进行原位扩增可以形成polonies(polymerase colonies),最后通过DNA测序进行分析。

SMI-seq方法能够精确定量多种蛋白,理论上这个阵列的密度可以达到每平方毫米一百万polonies。此外,那些共定位的polonies还揭示了蛋白质之间的互作。

为了证明这一技术的有效性,研究人员通过SMI-seq获得了G蛋白偶联受体和抗体结合的图谱。据介绍,SMI-seq是一种“一锅端”式的分析(one-pot assay),可以同时检测分子结合的亲和力和特异性。

研究显示,SMI-seq技术可以在单分子水平上原位检测蛋白及其复合体,从根本上提升蛋白质分析的灵敏度、准确性和多重性。该技术适用于二代测序平台,这进一步拓宽了它的应用范围。除了天然蛋白、重组蛋白,SMI-seq还可以用于人为生成的新蛋白、核酸和有条码的小分子。

著名遗传学George M. Church是哈佛医学院的遗传学教授、Wyss研究所的核心成员。他被誉为是个人基因组学和合成生物学的先锋。1984年,Church和Walter Gilbert发表了首个直接基因组测序方法,该文章中的一些策略现在仍应用在二代测序技术中。此外,如今的多重化分子技术和条码式标签也是他发明的。Church还是纳米孔测序技术的发明者之一。

原始出处:

Gu L, Li C, Aach J, Hill DE, Vidal M, Church GM.Multiplex single-molecule interaction profiling of DNA-barcoded proteins. Nature. 2014 Sep 21. doi: 10.1038/nature13761

Church GM在测序领域,合成生物学领域重要量论文:
Kosuri S, Church GM.Large-scale de novo DNA synthesis: technologies and applications. Nat Methods. 2014 May;11(5):499-507.

Lee JH, Daugharthy ER, Scheiman J, Kalhor R, Yang JL, Ferrante TC, Terry R, Jeanty SS, Li C, Amamoto R, Peters DT, Turczyk BM, Marblestone AH, Inverso SA, Bernard A, Mali P, Rios X, Aach J, Church GM.Highly multiplexed subcellular RNA sequencing in situ. Science. 2014 Mar 21;343(6177):1360-3.

Robasky K, Lewis NE, Church GM.The role of replicates for error mitigation in next-generation sequencing. Nat Rev Genet. 2014 Jan;15(1):56-62.

Lajoie MJ, Rovner AJ, Goodman DB, Aerni HR, Haimovich AD, Kuznetsov G, Mercer JA, Wang HH, Carr PA, Mosberg JA, Rohland N, Schultz PG, Jacobson JM, Rinehart J, Church GM, Isaacs FJ.Genomically recoded organisms expand biological functions. Science. 2013 Oct 18;342(6156):357-60

Goodman DB, Church GM, Kosuri S.Causes and effects of N-terminal codon bias in bacterial genes. Science. 2013 Oct 25;342(6157):475-9

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    2015-03-19 axin009
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    2015-04-09 liye789132251
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    2014-10-06 ylz8405

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