北京生物医学工程

一种基于反相微乳液的数字PCR微滴的制备方法

Preparation of droplets in digital PCR based on inverse microemulsion

作者：童石渊冯继宏白桦娟

单位：北京工业大学生命科学与生物工程学院(北京100124)

分类号：R318；Q781

出版年·卷·期（页码）：2017·36·5（517-519）

摘要：

目的提出一种新型的微滴生成方式——油包水(W/O)包裹技术，为数字PCR实验过程中微滴的制备提供一种更便捷的方式。方法通过配制一定比例的特定油相以及包含模板DNA等反应物的特定水相，在不同条件下形成W/O微滴，并进行PCR反应。结果所形成的微滴粒径大小均匀，并且在进行PCR反应过程中热力学性能保持不变，微滴不会因温度升降而破裂，适合于数字PCR中微滴的制备。结论生成的微滴可以有效地使模板DNA进行扩增反应。

Objective A new method for preparation of droplets is presented：wrapping technique for water-in-oil (W/O) ，it provides a more convenient way for the digital PCR experiment.Methods We formulated a certain percentage of particular oil phase with water phase containing the template DNA,maked the mixture to form W/O droplets,then carried out PCR reactions.Results The size of droplets prepared was uniform.Droplets’ thermodynamic properties remained constant during the PCR reactions,and the droplet didn’t rupture with the temperature changes.Conclusions The electrophoresis test results show that we make the amplification reaction of the template DNA successfully.

参考文献：

［1］Nakano M,Komatsu J,Matsuura SI,et al. Single-molecule PCR using water-in-oil emulsion［J］. Journal of Biotechnology,2003,102(2):117-124.

［2］Vogelstein B,Kinzler KW. Digital PCR［J］. Proceedings of the National Academy of Sciences of the United States of America,1999,96(16):9236-9241.

［3］Tawfik D,Griffiths A. Man-made cell-like compartments for molecular evolution［J］. Nature Biotechnology,1998,16(7):652-656.

［4］Dube S,Qin J,Ramakrishnan R. Mathematical analysis of copy number variation in a DNA sample using digital PCR on a nanofluidic device［J］. Plos One,2008,3(8):e2876.

［5］Kelley K,Cosman A,Belgrader P,et al. Detection of methicillin-resistant Staphylococcus aureus by a duplex droplet digital PCR assay［J］. Journal of Clinical Microbiology,2013,51(7):2033-2039.

［6］Robins HS,Ericson NG,Jamie G,et al. Digital genomic quantification of tumor-infiltrating lymphocytes［J］. Science Translational Medicine,2013,5(214):1102-1108.

［7］Phillip B,Tanner SC,Regan JF,et al. Droplet digital PCR measurement of HER2 copy number alteration in formalin-fixed paraffin-embedded breast carcinoma tissue［J］. Clinical Chemistry,2013,59(6):1155-1156.

［8］Schaerli Y,Wootton RC,Robinson T,et al. Continuous-flow polymerase chain reaction of single-copy DNA in microfluidic microdroplets［J］. Analytical Chemistry,2009,81(1):302-306.

［9］Hindson CM,Chevillet JR,Briggs HA,et al. Absolute quantification by droplet digital PCR versus analog real-time PCR［J］. Nature Methods,2013,10(10):1003-1005.

［10］Dingle TC,Sedlak RH,Cook L,et al. Tolerance of droplet-digital PCR vs real-time quantitative PCR to inhibitory substances［J］. Clinical Chemistry,2013,59(11):1670-1672.

［11］Schütze Tatjana,Rubelt F,Repkow J,et al. A streamlined protocol for emulsion polymerase chain reaction and subsequent purification［J］. Analytical Biochemistry,2011,410(1):155-157.

［12］Shi XL,Tang C,Wang W,et al. Digital quantification of gene expression using emulsion PCR［J］. Electrophoresis,2010,31(3):528-534.

［13］Leng X,Yang CJ. Agarose droplet microfluidics for highly parallel and efficient single molecule emulsion PCR［J］. Lab on a Chip,2010,10(21):2841-2843.

［14］Musyanovych A,Mailnder V,Landfester K. Miniemulsion droplets as single molecule nanoreactors for polymerase chain reaction［J］. Biomacromolecules,2005,6(4):1824-1828.

［15］Zhu Z,Zhang WH,Leng XF,et al. Highly sensitive and quantitative detection of rare pathogens through agarose droplet microfluidic emulsion PCR at the single-cell level［J］. Lab on a Chip,2012,12(20):3907-3913.

［16］Przybylski M,Dzieciatkowski T,Les K,et al. Comparison of real-time PCR quantitative analysis of the cytomegalovirus DNA level using LightCycler 2. 0 and LightCycler 480 instruments［J］. Journal of Clinical Virology,2012,55(3):270-273.

［17］Lina C,Nadeau JL. Uptake and processing of semiconductor quantum dots in living cells studied by fluorescence lifetime imaging microscopy (FLIM) ［J］. Chemical Communications,2013,49:1714-1716.

服务与反馈：

【文章下载】【加入收藏】

提示：您还未登录，请登录！点此登录