邻位连接技术（PLA）
在PLA技术中，不同类型的抗体上，连接有不同的核酸适体（1），另外二条寡核苷酸探针（2），各自都能与二个不同的核酸适体互补配对，在连接酶的作用下，二条寡核苷酸探针产生连接，因此再通过PCR扩增，没有读出DNA序列的要求，只是通过滚环复制产生“多连体”，再有荧光标记的单链核苷酸（3）与新合成的DNA序列配对，引入荧光标记，并且结合点众多，放大了靶点得荧光信号。不需要序列信息，只要有PCR扩增超长长度的产物就行。
换句话说，有无蛋白质相互作用，就看有无DNA连接反应发生。
火麻仁的作用二条寡核苷酸探针（2），各自都能与二个不同的核酸适体互补配对
DNA连接，之后是DNA复制（PCR反应）
通过滚环复制产生“多连体”
荧光标记的单链核苷酸（3）与新合成的DNA序列配对
大量的荧光标记杂交在一个靶点上
we decided to monitor and quantify the Dnmt1/PCNA interaction by proximity ligation in situ assay (P-LISA) in cells . As illustrated by the Figure 1A, the principle of this assay is bad on the staining of Dnmt1 and PCNA proteins by two antibodies, which are next revealed by condary antibodies conjugated with oligonucleotides. In pren入党考察情况
ce of hybridization solution and liga, the two oligonucleotides form with PLA a circle in ca of clo proximity of Dnmt1 and PCNA, here. Then, polymera and nucleotides participate to the formation of the rolling circle amplification, which are visualized in red fluorescence.洗浴策划
A
B
Figure 男生礼品1. Detection of endogenous Dnmt1/PCNA interactions using P-LISA.
(A) Schematic reprentation of the Dnmt1/PCNA proximity ligation in situ assay (P-LISA).
(B) Calibration of the microscopy (Axiovert 200 M Zeiss, Le Pecq, France) with ApoTome module. Left: picture of calibration performed with calibration balls (4 μm, Vue X-Y, Molecular Probes F36909). Right: picture of calibration performed with calibration balls (2.5 μm, Vue X-Z, Molecular Probes F36909). Blue and red were merged in lateral and axial.
硕士毕业论文致谢(C) Decovolving of picture realized with calibration balls (140 nm).
(D) Dnmt1/PCNA interactions were visualized in MCF10A cells. Red dots symbolize Dnmt1/PCNA interactions. Nucleus/DNA are stained in blue via the u of DAPI. Top left: ApoTome view, top right: orthogonal view, bottom: 3D views.
引自：Proximity ligation in situ assay for monitoring the global DNA methylation in cells.B
MC Biotechnology
Volume 11 2011.Eric Hervouet1,2 , Philippe Hulin2,3 , Fran?ois M Vallette炖牛肉怎么炖好吃1,2 and Pierre-Fran?ois Cartron1,2
PLA is an antibody-bad method in which either a single or two proteins (or antigens) are immunolabeled first with two primary antibodies and then with different species-specific condary antibodies conjugated to complementary oligonucleotides (8,9). When two antibody molecules are in clo proximity, the complementary DNA strands can be ligated, amplified, and visualized with a fluorescent probe as distinct puncta (Figure 1). Each spot may reprent a single complex containing each of two interacting proteins (or antigens). 特殊家庭The maximal distance between the condary antibodies in this assay is ~16 nm, only slightly larger than that for resonance energy transfer between fluorophores (~10 nm), the most common approach ud to infer GPCR oligomerization. By measuring clo proximity, PLA allows a validation in vivo of the molecular proximity of two endogenous proteins, something that cannot be est
ablished with simple colocalization studies, thereby making it possible to interrogate the existence and localization of interactions in vivo.
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